Novel manganese superoxide dismutase regulatory elements and uses therefore

ABSTRACT

A novel transcriptional regulatory element which was isolated from the MnSOD gene and which exhibits promoter-enhancer activity is disclosed. The promoter-enhancer activity of the element is further modulated by inflammatory mediators to regulate transcription. Methods of using the promoter-enhancer element to regulate gene expression, and therapeutic uses involving the promoter-enhancer element are also described.

RELATED APPLICATIONS

This application is a Divisional Application of application Ser. No.09/856,766, filed on Aug. 28, 2001, which is a § 371 Application ofPCT/US99/2833 1, filed on Nov. 30, 1999, which claims priority to U.S.Ser. No. 60/110,334, filed on Nov. 30, 1998. The contents of all of theaforementioned application(s) are hereby incorporated herein in theirentirety by this reference.

GOVERNMENT FUNDING

This invention was made with government support under grant HL-39593awarded by the National Institutes of Health. The United Statesgovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to novel transcriptional regulatory elementsderived from manganese superoxide dismutase (MnSOD) genes, as well asmethods of identifying and using the regulatory elements to control geneexpression.

BACKGROUND OF THE INVENTION

Precise control of regulated gene expression has multiple potentialapplications including inducible gene targeting, overexpression ofcytotoxic or cytoprotective genes, antisense RNA expression, and somaticgene therapy (Wettstein et al., 1988).

The ability to produce biologically active polypeptides is increasinglyimportant to the pharmaceutical industry. Over the last decade, advancesin biotechnology have led to the production of important proteins andfactors from bacteria, yeast, insect cells and from mammalian cellculture. Mammalian cultures have advantages over cultures derived fromthe less advanced life forms in their ability to post-translationallyprocess complex protein structures such as disulfide-dependent foldingand glycosylation. Neuroendocrine cell types have added uniquecapacities of endoproteolytic cleaving, C-terminal amidation andregulated secretion. Indeed, mammalian cell culture is now a preferredsource of a number of important proteins for use in human and animalmedicine, especially those which are relatively large, complex orglycosylated. Improved methods for expressing desirable polypeptides inmammalian host cells are highly desirable.

Gene therapy involves the transfer of one or more functional homologousgenes, and the sequences controlling their expression, into a targetcell. The purpose of gene therapy is to replace a defective or deficientgene, the absence of which produces a pathological state or tosupplement an endogenous gene product to achieve a therapeutic effect(Berns and Giraud, 1995). Viral vectors are widely used vehicles for theeffective delivery of genes into mammalian cells which have thecapability to infect high proportions of cells in a cell population(Friedmann and Yee, 1995; Friedmann, 1997). Some of the best examples ofviral gene targeting vectors are based on retroviral, adeno (Ad) oradeno-associated (AAV) viruses. However, vectors developed from theseviruses all lack some level of specificity which presents an obstaclefor appropriate and controlled expression of foreign genes (Friedmann,1996). For example, retroviruses are generally limited to transductionof dividing cells whereas Ad and AAV can transduce non-dividing cells.On the other hand, repeated administration of recombinant Ad basedvectors, is often limited by host immune responses against viralstructural proteins. Presently, AAV may hold the greatest promise inthat rAAV does not appear to induce an inflammatory or immune response,and is only limited by the inability to easily produce high rAAV viriontiters.

AAV is a single stranded human DNA virus with a genome length of 4.7 kb(Muzyczka, 1992; Srivastava et al., 1983; Yang and Trempe, 1993) thatrequires a helper virus for productive growth. Adeno (Ad) or herpesvirus family members can provide helper function in established humantissue culture lines, whereas only adenovirus is found associated withAAV in human isolates. The normal route of infection for AAV is via therespiratory or intestinal tracts analogous to Ad. If a helper virus isnot available during AAV infection, the AAV genome integrates into ahuman chromosome 19 and is propagated as a stable provirus.Superinfection with Ad leads to AAV provirus excision and a normalproductive growth cycle that results in the production of a mixed viralstock consisting of both wild type AAV and Ad virus particles. AAVpossesses unique biological properties, which has led to itsexploitation as a versatile gene therapy vector. Most notably AAVundergoes a latency phase, which often involves stable integrationwithin a region of chromosome 19 known as the AAVS1 site, thusestablishing a persistent infection with very little host response(Cheung et al., 1980; Conrad et al., 1996). Perhaps the most attractiveAAV feature is that even though human exposure to this virus iscommonplace, no diseases has been associated with AAV infections ineither animal or human populations (Blacklow et al., 1967; Blacklow etal., 1968; Blacklow et al., 1971; Hoggan, 1970), nor have there been anyreports of rAAV induced inflammatory responses. In addition, it has beendemonstrated that recombinant AAV (rAAV) vectors do not integrate inlung epithelial cells (Flotte et al., 1993; Afione et al., 1996). rAAVpersists for up to six months potentially as an unintegrated episome.

Numerous AAV vectors have been developed to exploit the latency pathway,where, in general, vectors are generated by deleting the viral codingsequences and substituting the appropriate transgene controlled by apromoter and flanked by the AAV-TRs. By keeping the construct size at ˜5kb which is within the packaging limit, these vectors can beincorporated into infectious virions in trans in Ad-infected cells. Suchrecombinant virions have been found to infect a variety of cell types invitro and in vivo including hematopoietic cells (Goodman et al., 1994)neurons (Kaplitt et al., 1994), airway epithelial cells (Flotte et al.,1992), as well as skeletal and cardiac muscle (Kessler et al., 1996).

One of the main issues in potential clinical application of gene therapyis the need for increased gene transfer efficiency and targetspecificity associated with regulated expression at therapeuticallyrelevant levels in vivo (Chow et al., 1997). Effective gene therapy,therefore, must include the design of crucial regulatory elements,promoters and enhancers, which possess cell type specific activities andcan be activated by certain physiologically relevant induction factors(e.g., hormones, cytokines, chemokines, irradiation, heat shock) viaresponsive elements. Controlled and restricted expression can beachieved using such regulatory elements to drive the expression oftherapeutic genes in plasmid based as well as viral vector constructs(Hwang et al., 1997; Sandig and Strauss, 1996; Finke et al., 1998). Inaddition to high level and efficient gene expression, minimizing orexcluding inappropriate gene expression in surrounding non-target cellswill be of great importance for numerous gene therapy applications(Namba et al., 1998; Hesdorffer et al., 1998; Gossen and Bujard, 1992).

Unfortunately, almost all of the presently available inducible promotersused in gene therapy vectors require exogenous stimulation bynon-physiological or artificial substances such as tetracycline (No etal., 1996), ecdysone (Delort and Capecchi, 1996), or RU486 (Massie etal., 1998). One of the disadvantages of these systems is that they allrequire expression of two gene products. Namely, the expression of thedesired transgene driven by an inducible promoter which requires theexpression of a non-endogenous receptor. This is usually accomplished byco-transfection of separate plasmids into mammalian cells, thuspotentially limiting the size of the transgene when a viral vectorsystem is used for transgene delivery. In addition, to maintain highlevels of expression with the aforementioned inducible promoters, theexogenous substance must be continuously supplied. The disadvantages ofthese systems include the continuous treatment with the exogenoussubstance and slow clearance from the organism, which interferes withquick and precise induction. Alternatively high levels of constitutiveexpression can be obtained with the cytomegalovirus (CMV) promoter inmany cell types, which will be beneficial in certain disease states,where an example might be the CFTR gene product for cystic fibrosis.

It has been suggested that the enhancer elements of mammalian genesmight allow the desired precise control of gene regulation needed fortransgene expression (Maxwell et al., 1996; Clesham et al., 1996;Walther and Stein, 1996; Hofmann et al., 1996; Raoul et al., 1998). Suchan enhancer element should be tissue-specific and stimulant-specific,allowing the transgene expression to be kept at a low basal level.However, such an element should allow dramatic induction in response tothe precise stimulus. Ideally, the stimulus causing this induction couldbe endogenously produced and potentially associated with the diseasepathology. Most intracellular trans-activators of enhancer elements aretypically stimulated as part of signal transduction pathways fromtransmembrane or intracellular receptors responding to extracellularligands (hormones, cytokines, chemokines). Therefore, unlike thepresently available inducible promoters systems, an ideal regulatorysequence would function through an endogenous stimulus, receptor andsignal transduction pathway.

Many diseases amenable to gene therapy will not require continuous highconstitutive gene expression since the disease state itself is episodicin nature (inflammatory or ischemic diseases). In these situations, lowlevels of gene expression during the normal state are adequate, but thecapacity to increase expression during the onset of inflammation orischemia would be advantageous for the precise control sought. Highlevel expression of cytoprotective proteins (such as MnSOD, Hohmeier etal., 1998; Majima et al., 1998; Epperly et al., 1998) duringinflammation/ischemia of various organ systems (lungs, brain, small andlarge intestine) would either halt or slow progression of the diseaseprocess (Waxman et al., 1998; Manna et al., 1998; Arai et al., 1990).However, available inducible promoters do not allow the cell to controlthe timing of expression of the transgene or its fold induction. Anideal inducible element would make use of the cell or tissues ownsignaling system to increase expression of the transgene of interest.Under conditions of inflammation, the most precise inducible elementwould probably utilize the cytokine systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic representation of the promoter deletions ofthe 5′ flanking sequence of the rat MnSOD gene. The first exon isdepicted by a box, and numbers indicate the position of the restrictionenzyme sites relative to the first ATG signal. Placement of the deletionfragments 5′ to the human growth hormone reporter gene in thepromoter-less human growth hormone vector (pØGH) is indicated.

FIG. 1B shows a Northern analysis of hGH and MnSOD mRNA levels in ratlung epithelial cells transfected with pØGH vectors containing promoterdeletion fragments of the rat MnSOD gene (FIG. 1A), and stimulated withLPS.

FIG. 1C shows levels of human growth hormone (hGH) protein expressed inrat lung epithelial cells transfected with pØGH vectors containingpromoter deletion fragments of the rat MnSOD gene (FIG. 1A), andstimulated with LPS, TBF-α, or IL-1β. Error bars represent the standarderror of the mean. A p value of <0.05 was considered significant and isindicated by the *.

FIG. 2A shows a schematic representation and restriction map of a ratMnSOD genomic clone, and the construction of pØGH expression vectorscontaining restriction fragments of the MnSOD gene to assess potentialenhancer activity. The restriction enzyme sites are indicated above thesequence. DNase I hypersensitive sites 2 through 7 (★) are alsoindicated.

FIG. 2B shows a Northern analysis of hGH and MnSOD mRNA levels in ratlung epithelial cells transfected with the pØGH vector containing the6.1 Kb internal Hind III fragment of the rat MnSOD gene (FIG. 2A), andstimulated with LPS.

FIG. 2C shows a Northern analysis of hGH and MnSOD mRNA levels in ratlung epithelial cells transfected with pØGH vectors containing eitherthe 3.8 Kb or the 2.3 Kb internal fragments of the rat MnSOD gene (FIG.2A), and stimulated with LPS.

FIG. 3A shows a schematic representation of serial deletions of the 3.8Kb internal enhancer fragment of the rate MnSOD gene.

FIGS. 3B and 3C show a Northern analysis of hGH and MnSOD mRNA levels inrat lung epithelial cells transfected with pØGH vectors containing 3′deletion fragments of the 3.8 Kb internal enhancer fragment of the ratMnSOD gene, and stimulated with LPS.

FIGS. 3D and 3E show a Northern analysis of hGH mRNA levels in rat lungepithelial cells transfected with pØGH vectors containing 5′ deletionfragments of the 3.8 Kb internal enhancer fragment of the rat MnSODgene, and stimulated with LPS.

FIG. 4A shows a schematic representation of a 260 bp enhancer region inintron 2 of the rat MnSOD gene, and internal fragments of the enhancerregion generated for transfection studies and electrophoretic mobilityshift assays (EMSA). Putative constitutive and inducible protein bindingsites are illustrated by open circles (∘) and filled triangles (▴),respectively.

FIG. 4B shows a Northern analysis of hGH mRNA levels in rat lungepithelial cells transfected with the pØGH vector containing either the919 bp (FIG. 3A) or the 260 bp fragment (FIG. 4A) of the MnSOD enhancerregion, and stimulated with LPS, TBF-α, or IL-1β.

FIG. 4C shows a Northern analysis of hGH mRNA levels in rat lungepithelial cells transfected with the pØGH vector containing either the260 bp MnSOD enhancer region, or the internal 143 bp fragments (FIG. 4A)of the 260 bp MnSOD enhancer, and stimulated with LPS.

FIGS. 4D and 4E show an EMSA analysis of protein binding within theMnSOD enhancer region. Internal fragments of the 260 bp enhancer regionof the rat MnSOD gene (FIG. 4A) were used as probes, and incubated withnuclear extracts from untreated cells and cells treated with LPS, TBF-α,and IL-1β. DNA-protein complexes are indicated by arrowheads (

).

FIG. 5 shows an alignment of the rat MnSOD enhancer region with theanalogous region of intron 2 of the human MnSOD gene.

FIG. 6A shows a schematic representation indicating the placement of therat and human MnSOD enhancer fragments 5′ to the human growth hormonereporter gene in the thymidine kinase promoter-human growth hormonevector (pTKGH).

FIG. 6B shows a Northern analysis of hGH and MnSOD mRNA levels in ratlung epithelial cells transfected with the pTKGH vector containing the6.1 Kb internal Hind III fragment of the rat MnSOD gene (FIG. 2A), andstimulated with LPS, TBF-α, or IL-1β.

FIG. 6C shows a Northern analysis of hGH mRNA levels in rat lungepithelial cells transfected with the pTKGH vector containing either a553 bp fragment containing the rat MnSOD enhancer, or a 466 bp fragmentcontaining the human MnSOD enhancer, and stimulated with LPS, TBF-α, orIL-1β.

FIG. 7 shows a Northern analysis of hGH and MnSOD mRNA levels in ratintestinal epithelial cells transfected with the pTKGH vector containinga 508 bp fragment containing the MnSOD enhancer.

FIG. 8A shows a schematic representation of deletion fragments of therat MnSOD promoter, indicating protein binding sites found by in vivofootprinting (●). Placement of the promoter deletion fragments 5′ to thehuman growth hormone reporter gene in the promoterless human growthhormone vector (pØGH) containing the rat MnSOD enhancer fragment isindicated.

FIGS. 8B and 8C show a Northern analysis of hGH mRNA levels in rat lungepithelial cells transfected with the pØGH vector containing the ratMnSOD enhancer and promoter deletion fragments of the rat MnSOD gene(FIG. 6A), and stimulated with LPS.

FIG. 9A shows a schematic representation of the promoter-less humangrowth hormone vector (pØGH) and the position of the human 466 bp MnSODprohancer fragment. Northern analysis of hGH mRNA levels in rat lungepithelial cells transfected with the pØGH vector containing the human466 bp MnSOD prohancer, and stimulated with LPS, TBF-α, or IL-1β.

FIG. 9B shows a schematic representation of the promoter-less humangrowth hormone vector (pØGH) and the position of the rat 746 bp, 553 bpand 260 bp MnSOD prohancer fragments. Northern analysis of hGH mRNAlevels in rat lung epithelial cells transfected with the pØGH vectorcontaining the rat MnSOD prohancer fragments, and stimulated with LPS,TBF-α, or IL-1β.

FIG. 10 shows restriction maps of the pTR-UF (User Friendly) series ofAAV vector plasmids constructed by the Vector Core at the University ofFlorida. pTR-UF2 and pTR-UF3 contain the jellyfish gfp cDNA sequencewith a humanized codon preference. pTR-UF3 is identical to pTR-UF2except that pTR-UF3 has a poliovirus IRES element that allows theconstruction of bicistronic genes.

FIG. 11 shows AAV vectors developed for gene targeting of CFTR.

FIG. 12 shows regulation of the MnSOD gene by inflammatory mediators invarious cell types.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated polynucleotidecomprising a manganese superoxide dismutase (MnSOD) regulatory elementcapable of causing inducible transcription or expression of an operablylinked heterologous polynucleotide. In one embodiment, the regulatoryelement is derived from intron 2 of the rat MnSOD gene and, inparticular, the region of intron 2 having the nucleotide sequence shownin SEQ ID NO: 1. In a specific embodiment, the regulatory elementcomprises the 260 nucleotides from SEQ ID NO: 1 shown in SEQ ID NO:5. Inanother embodiment, the element is derived from intron 2 of the humanMnSOD gene and, in particular, the region of intron 2 having thenucleotide sequence shown in SEQ ID NO:2.

The invention also encompasses highly homologous (e.g., having at leastabout 60% sequence identity) regulatory elements from other mammalianMnSOD genes which also are capable of causing inducible transcription orexpression of an operably linked heterologous polynucleotide. Inparticular embodiments, the invention encompasses an MnSOD regulatoryelement comprising a nucleotide sequence which is at least about 70%identical to SEQ ID NO:1 or at least about 90% identical to SEQ ID NO:2.

In another aspect, the invention provides an isolated manganesesuperoxide dismutase regulatory element operably linked to aheterologous polynucleotide such that, upon activation of the regulatoryelement, transcription or expression of the heterologous polynucleotideis induced. In one embodiment, the regulatory element is activated(i.e., induces transcription or expression of an operatively linkedheterologous polynucleotide) in the presence of an inflammatorystimulus, such as TBF-α, IL-1β or LPS. In another embodiment, theregulatory element is activated in the presence of 5-aminosalicylicacid.

The heterologous polynucleotide can be any polynucleotide capable ofbeing transcribed or expressed, such as a gene encoding a protein orpolypeptide (e.g., a cytoprotectant) or a polynucleotide which istranscribed as an antisense mRNA. The invention also provides a cell(e.g., a mammalian cell) transformed either in vivo or in vitro with anisolated MnSOD regulatory element of the invention.

In another aspect, the invention provides an inducible expression systemcomprising (a) an isolated MnSOD regulatory element of the inventionwhich induces transcription or expression of an operably linkedheterologous polynucleotide upon activation; and (b) a compound whichactivates the regulatory element, or a polynucleotide encoding acompound which activates the regulatory element.

The inducible expression system can also include a heterologouspolynucleotide operably linked to the MnSOD regulatory element suchthat, upon activation of the MnSOD regulatory element by the compound,transcription or expression of the heterologous polynucleotide isinduced. The inducible expression system can further include aheterologous promoter (i.e., a promoter which is not derived from theMnSOD regulatory element itself) to further increasetranscription/expression levels of the heterologous polynucleotide.

Accordingly, in yet another aspect, the invention provides a method forachieving inducible transcription or expression of a heterologouspolynucleotide in a cell by introducing the above-described MnSODregulatory element or expression system into a cell under conditionssuitable for transcription and/or expression of a heterologouspolynucleotide. The method can further include the step of introducinginto the cell an effective amount of a compound which activates theregulatory element to induce transcription or expression of anoperatively linked polynucleotide, or a polynucleotide encoding thecompound.

Other aspects and embodiments of the invention shall be apparent fromthe following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification and isolation of anovel inducible MnSOD transcriptional regulatory element having dualpromoter-enhancer (“prohancer”) functions. The inducible prohancerelement can be regulated by inflammatory mediators, (e.g.,lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α),interleukin-1beta (IL-1β)), as well as other compounds, for the controlof gene expression.

DEFINITIONS

Before further description of the invention, certain terms employed inthe specification, examples, and appended claims are defined below.

An “isolated polynucleotide” or “isolated nucleic acid molecule” refersto a polynucleotide (e.g., DNA, RNA) which is removed from its naturalsequence context. For example, with regards to genomic DNA, the term“isolated” includes nucleic acid molecules which are separated from thechromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. The isolated polynucleotide can beany polynucleotide that is capable of being transcribed or translated ina cell. The isolated polynucleotide (genomic or cDNA clone) can be, forexample, cloned into a vector. Except as noted hereinafter, standardtechniques for cloning, DNA isolation, amplification and purification,for enzymatic reactions involving DNA ligase, polymerase, restrictionendonucleases and the like and various separation techniques are thoseknown and commonly employed by those skilled in the art. A number ofstandard techniques are described in: Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wu(ed.)(1979) Meth. Enzymol 68; Wu et al. (Eds.) (1983) Meth. Enzymol. 100& 101; Grossman and Moldave (eds.) (1980) Meth. Enzymol. 65; Miller (ed)(1972) Exp. Mol. Genetics, Cold Spring Harbor, N.Y.; Old and Primrose(1981) Principles of Gene Manipulation, Univ. of Cal. Press, Berkeley;Schlief and Wensink (1982) Practical Methods in Molecular Biology;Glover (ed) 1985 (DNA Cloning, Vols. I and II, IRL Press, Oxford, UK;Sellow and Hollaender (1979) Genetic Engineering: Principles andMethods, Vols I, Plenum Press, NY; which are incorporated by referencein their entirety herein. Abbreviations, where employed, are thosedeemed standard in the field and commonly used in professional journalssuch as those cited herein.

As used herein, the term “nucleic acid molecule” or “polynucleotide” isintended to include DNA molecules (e.g., cDNA or genomic DNA) and RNAmolecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “derived from”, as used herein, refers to an actual ortheoretical source or origin for isolated polynucleotides of theinvention. For example, a polynucleotide that is “derived from” aparticular polynucleotide (e.g., a MnSOD gene) will be identical orhighly homologous in nucleotide sequence to a relevant portion of thereference polynucleotide (e.g., a MnSOD gene). Thus, for example, apolynucleotide that is “derived from” the intronic sequences (e.g.,intron 2) of a MnSOD gene may correspond in nucleotide sequence to allor a portion of the intronic sequences of a wild-type MnSOD gene.Isolated polynucleotides of the invention which are “derived from” MnSODgenes (e.g., intron 2) also include those which have been modified byinsertion, deletion or substitution of one or more nucleotides but whichretain substantially the same activity or function.

A DNA “coding sequence”, “coding region”, or a “sequence encoding” aparticular protein is a DNA sequence which is transcribed and translatedinto a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory elements. The boundaries of the coding sequenceare determined by a start codon at the 5′-terminus and a translationstop codon at the 3′-terminus. A coding sequence can include, but is notlimited to, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian, animal, avian, etc.) or prokaryoticsources, as well as synthetic DNA sequences. A transcription terminationsequence will usually be located 3′ to the coding sequence.

A “MnSOD gene”, as used herein, refers to a MnSOD gene (e.g., a clonedgenomic gene or a cDNA), including its untranscribed upstream anddownstream regions and transcribed, untranslated regions from anyspecies which naturally expresses MnSOD. The nucleotide sequence for thegenomic human MnSOD gene is available at GenBank Accession No. S77127. A“nonhuman MnSOD gene”, as used herein, refers to a MnSOD gene (e.g., acloned genomic gene or a cDNA), including its untranslated regulatoryregions, from any species excepting human (e.g., rat, mouse, avian,sheep, porcine, bovine). For example, the nucleotide sequence for therat MnSOD is available at GenBank Accession No. X56600.

The terms “regulatory element”, “control element” and “regulatorysequence” are used interchangeably herein, and refer to a nucleic acidwhich, when operably linked to a polynucleotide, modulates transcriptionand/or expression levels of the polynucleotide in a cell. Geneticregulatory elements of the present invention may include promoters,enhancers, or a combination thereof, as well as other cis-actingsequences involved in the binding of transcription factors. Regulatoryelements include both positive and negative regulators of transcription.

The term “promoter”, as used herein, refers to a DNA sequence which isgenerally placed adjacent to the 5′ end of a structural gene, locatedproximal to the start codon, that is involved in the initiation oftranscription of the adjacent gene. Promoters contain DNA sequenceelements that mediate the binding of an RNA polymerase, and modulate thelevel of transcription. Further, specific regulatory sequences within oradjacent to promoters that are functional in the regulation (inductionand repression) of gene expression responsive to stimuli or specificchemical species may also be present. If a promoter is “inducible”, thenthe rate of transcription increases in response to an inducing agent or“inducer”.

The term “enhancer”, as used herein, refers to a regulatory sequence,which can function in either orientation and in any location withrespect to a promoter, to modulate (e.g., increase) the effect of apromoter (e.g., to increase transcription levels). For example, anenhancer of the present invention may act in a position-independent andan orientation-independent manner to induce transcription of anoperatively linked polynucleotide.

The terms “promoter-enhancer” or “prohancer” are used interchangeablyherein, and refer to an inducible regulatory element capable ofinitiating transcription and/or expression of a polynucleotide sequenceto which it is operatively linked. A prohancer element can actindependently of a classical promoter to initiate and regulatetranscription when it is operatively linked upstream (e.g., 5′) of agene, thus acting as a promoter. A prohancer element can also functionas an enhancer, acting in a position and orientation independent fashionin conjunction with a promoter (e.g., herpes virus TK promoter) toregulate inducible gene transcription (e.g., under conditions ofinflammation).

The terms “inducible transcription” or “induction of transcription” areused interchangeably herein, and refer to the initiation oftranscription by interaction of an “inducer” with a transcriptionalregulatory protein and/or element. An “inducer” includes, but is notlimited to, any protein, polypeptide, nucleic acid, carbohydrate, smallmolecule, or stimulus that, either directly or indirectly, triggers genetranscription by binding to a transcriptional regulatory protein and/orelement. For example, gene expression is regulated by factors such asenvironmental factors (e.g., temperature, light, and oxygen tension),chemical species (e.g., nutrients, metabolites, heavy metal ions),cytokines and steroids. The exact mechanism of regulation by suchsignals or stimuli is likely to be complex, involving multiple proteininteractions. By analogy to previous mechanistic studies oftranscriptional regulation, however, regulatory control is expected toinvolve changing the ability of RNA polymerase to bind to DNA sequencesin the promoter region. Suitable inducers of MnSOD prohancer elements ofthe invention include inflammatory mediatiors (e.g., LPS, TNF-α andIL-1β) and benzene derivatives, e.g., 5-aminosalicylic acid.

“Expression” of a gene requires both transcription of DNA into mRNA, andthe subsequent translation of the mRNA into protein products.

As used herein, the term “heterologous” is defined in relation to apredetermined reference polynucleotide sequence, and includes anynon-identical polynucleotide, or any polynucleotide that does notnaturally occur adjacent to the reference sequence. For example, withrespect to a reference promoter sequence, a heterologous polynucleotideincludes any polynucleotide that is not identical to the promoter towhich it is operatively linked.

The term “operatively linked” or “operably linked” is intended to meanthat molecules are functionally coupled to each other in that the changeof activity or state of one molecule is affected by the activity orstate of the other molecule. Nucleotide sequences are “operably linked”when a transcriptional regulatory sequence functionally relates to theDNA sequence encoding the polypeptide, protein, or antisense mRNA ofinterest. For example, a promoter nucleotide sequence is operably linkedto a DNA sequence encoding a protein, polypeptide, or antisense mRNA ofinterest if the promoter nucleotide sequence controls the transcriptionof the DNA sequence encoding the protein or mRNA of interest. Typically,two polypeptides that are operatively linked are covalently attachedthrough peptide bonds.

The term “reporter gene”, as used herein, refers to a gene encoding aprotein which is readily quantifiable or observable. Becausegene,regulation usually occurs at the level of transcription,transcriptional regulation and promoter activity are often assayed byquantitation of gene products. For example, promoter regulation andactivity has often been quantitatively studied by the fusion of theeasily assayable E. coli lacZ gene to heterologous promoters (Casadabanand Cohen (1980) J. Mol. Biol. 138:179-207). The structural gene forchloramphenicol acetyl transferase (CAT), human growth hormone (hGH),and green fluorescence protein (GFP) are other genes commonly used todetect activity of a promoter or other regulatory sequence.

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. As used herein, the term vector is intended toinclude a nucleic acid molecule capable of transporting another nucleicacid to which it has been linked. A vector may be characterized by oneor a small number of restriction endonuclease sites at which such DNAsequences may be cut in a determinable fashion without the loss of anessential biological function of the vector, and into which a DNAfragment may be spliced in order to bring about its replication andcloning. A vector may further contain a marker suitable for use in theidentification of cells transformed with the vector. One type of vectoris a “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. However, the invention isintended to include other forms of expression vectors, such as viralvectors (e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

An “expression vector” means any DNA vector (e.g., a plasmid vector)containing the necessary genetic elements for expression of a desiredgene, including a promoter region of the present invention. Theseelements are “operably linked” to the gene, meaning that they arelocated at a position within the vector which enables them to have afunctional effect on transcription of the gene. The regulatory elementsneed not be contiguous with the coding sequence, so long as theyfunction to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter and the coding sequence and the promoter can still beconsidered “operably linked” or “in operable linkage to” the codingsequence.

A cell has been “transformed” by exogenous DNA (e.g., a transgene) whensuch exogenous DNA has been introduced inside the cell membrane.Exogenous DNA may or may not be integrated into the chromosomal DNAcomprising the genome of the cell. With respect to eukaryotic cells, astably transformed cell is one in which the exogenous DNA has becomeintegrated into the chromosome such that it is inherited by daughtercells though chromosome replication.

A “cell” or “host cell” of the invention includes any cell that can bemodified by the introduction of heterologous DNA. A host cell of thepresent invention includes prokaryotic cells and eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample, E. Coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. Eukaryotic cells include,but are not limited to, yeast cells, plant cells, fungal cells, insectcells (e.g., baculovirus), mammalian cells, and the cells of parasiticorganisms, e.g., trypanosomes.

As used herein, the term “yeast” includes not only yeast in a stricttaxonomic sense, i.e., unicellular organisms, but also yeast-likemulticellular fungi of filamentous fungi. Exemplary species includeKluyverei lactis, Schizosaccharomyces pombe, Ustilaqo maydis, andSaccharomyces cerevisiae. Other yeast which can be used in practicingthe present invention are Neurospora crassa, Aspergillus niger,Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenulapolymorpha.

Mammalian host cell culture systems include established cell lines suchas COS cells, L cells, 3T3 cells, Chinese hamster ovary (CHO) cells,embryonic stem cells, and HeLa cells.

A “transgene” refers to a nucleic acid which is introduced into a cell.Typically, the transgene is integrated into the genome of the cell bygene targeting or homologous recombination. The transgene can encode aprotein which is not expressed in the cell or which is expressed in thecell at low levels or in defective form.

A “transgenic animal” is an animal carrying in its cells at least onetransgene which has been introduced into the germline of the animal,such that the introduced gene is present in all somatic and germlinecells. A transgenic animal can contain a transgene that is eitherhomologously or non-homologously integrated into an endogenouschromosomal location (Hogan et al. (1994) Manipulating the Mouse Embryo,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y).

I. MnSOD Prohancer Elements

Novel MnSOD transcriptional regulatory elements or “prohancers” of theinvention can act as a promoter and/or an enhancer when operatively to aheterologous polynucleotide sequence. In the absence of stimulus,negligible levels of transcription occur, but transcription issignificantly increased in the presence of an inducer (e.g., aninflammatory mediator or 5-aminosalicylic acid).

MnSOD prohancer elements of the invention can be located within intron 2of the MnSOD gene and are typically conserved among different species.The element acts as a true enhancer of gene transcription in response toinflammatory mediators (e.g., lipopolysaccharide (LPS), tumor necrosisfactor alpha (TNF-α), interleukin-1 beta (IL-1β)). However, a novelproperty of the element is that it can additionally function in theabsence of a promoter to stimulate gene transcription of a heterologousgene. Thus, the element has been designated a “prohancer” to signifythat it is a true enhancer which can promote transcription in theabsence of a true promoter, most importantly, under physiologicconditions of inflammation.

Accordingly, one aspect of the invention pertains to an isolatedpolynucleotide which includes an MnSOD prohancer element, orbiologically active portion thereof, as well as nucleic acidhybridization probes which can be used to identify MnSOD prohancerelements (e.g., used as PCR primers for the amplification or mutation ofMnSOD prohancer elements).

Novel MnSOD prohancer elements of the invention (e.g., comprising all ora portion of the nucleotide sequence shown in SEQ ID NO:1 or 2) can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. For example, using all or portion of thenucleic acid sequence of SEQ ID NO:1 or 2 as a hybridization probe,MnSOD prohancer elements can be isolated using standard hybridizationand cloning techniques (e.g., as described in Sambrook, J., Fritsh, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Prohancer elements comprising all or a portion of SEQ ID NO:1 or 2 alsocan be isolated by polymerase chain reaction (PCR) using syntheticoligonucleotide primers corresponding to all or a portion of thesequences of SEQ ID NO:1 and 2. For example, prohancer elements can beamplified using cDNA, mRNA or, alternatively, genomic DNA, as a templateand appropriate oligonucleotide primers according to standard PCRamplification techniques. The nucleic acid so amplified can be clonedinto an appropriate vector and characterized by DNA sequence analysis.Oligonucleotides corresponding to MnSOD prohancer elements nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In one embodiment, an isolated prohancer element of the invention isderived from (i.e., comprises all or a portion of ) the nucleotidesequence set forth in SEQ ID NO:1 or the complement thereof,corresponding to a portion of intron 2 of the rat MnSOD gene. Within thesequence of SEQ ID NO:1, a minimal effective promoter element has beenmapped to a region of about 260 base pairs shown in SEQ ID NO:5.

In another embodiment, an isolated prohancer element of the invention isderived from the nucleotide sequence set forth in SEQ ID NO:2 or thecomplement thereof, corresponding to a portion of intron 2 of the humanMnSOD gene. Minimal effective promoter elements within SEQ ID NO:2 canbe mapped in the same manner as is described herein for SEQ ID NO:1.

Isolated regulatory elements having the biological activity of an MnSODprohancer element generally consist of at least about 230, 230-260, 260,260-300, 300-350, 350 or more nucleotides in length and can be derivedfrom SEQ ID NO:1 or SEQ ID NO:2. For example, the 260 bp element shownin SEQ ID NO:5 corresponds to a prohancer element derived from SEQ IDNO:1 (rat).

The nucleotide sequences provided in SEQ ID NOS: 1 and 2 also allow forthe generation of probes and primers which can be used to identifyand/or clone MnSOD prohancer elements from other species. Theprobe/primer typically comprises substantially purified oligonucleotide.Probes based on the provided MnSOD prohancer nucleotide sequences can beused to detect transcripts or genomic sequences encoding the same orrelated prohancer elements. Thus, the probe can also contain a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Suitableoligonucleotide probes typically comprise a region of nucleotidesequence that hybridizes under stringent conditions to at least about 12or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45,or 50 consecutive nucleotides of a sense sequence of SEQ ID NO:1 or 2,of an anti-sense sequence of SEQ ID NO:1 or 2, or of a naturallyoccurring variant or mutant of SEQ ID NO:1 or 2.

Accordingly, proenhancer elements of the invention also includeregulatory sequences which are highly homologous (i.e., which share atleast about 60%, preferably at least about 70%, and more preferablyabout 80-90% or more sequence identity) with all or a biologicallyactive portion of SEQ ID NOS:1 and 2. In a particular embodiment, theprohancer element comprises a nucleic acid sequence having at leastabout 90%, 95%, 98%, or more identity with the sequence of SEQ ID NO:1.In another particular embodiment, the prohancer element comprises anucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%,95%, or more identity with the sequence of SEQ ID NO:2. The percentidentity to the prohancer sequence of either SEQ ID NO:1 or SEQ ID NO:2need not be limited to the specific percentages given, but is also meantto include all sequences derived from the nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:2, which retain prohancer function, e.g., the abilityto promote inducible transcription of a polynucleotide operativelylinked to the prohancer element.

Highly homologous nucleic acid molecules (e.g., corresponding tonaturally occurring variants of the provided MnSOD prohancer elementsand/or functionally related prohancer elements) can be isolated based ontheir homology to the MnSOD prohancer elements disclosed herein usingthe polynucleotide sequences disclosed herein, or a portion thereof, asa hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions. For example, nucleic acidmolecules corresponding to related MnSOD prohancer elements can bemapped to the same intronic locus (e.g., intron 2) within MnSOD genesfrom species other than rat and human.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1 or 2. In anotherembodiment, a polynucleotide of the present invention comprises anucleotide sequence which is at least about 150, 200, 250, 260, 300,350, 400, 450 or more nucleotides in length and hybridizes understringent hybridization conditions to the nucleotide sequence of SEQ IDNO:1 or 2, and has prohancer function. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% identical to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 70%, more preferably at least about 80%, evenmore preferably at least about 85% or 90% identical to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 60° C., and even morepreferably at 65° C. Preferably, an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequenceof SEQ ID NO:1 or 2 corresponds to a naturally-occurring nucleic acidmolecule. As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature.

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least 70%, 80%, 90%or 95% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein nucleic acid “identity” isequivalent to nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the percent identity between twonucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1989) which has been incorporated into theALIGN program (version 2.0) (available athttp://vega.igh.cnrs.fr/bin/align-guess.cgi).

Nucleic acid sequences can further be used as a “query sequence” toperform a search against public databases to, for example, identifyother family members or related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain homologous nucleotide sequences. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., NBLAST) can beused. See http://www.ncbi.nlm.nih.gov. Additionally, the “Clustal”method (Higgins and Sharp, Gene, 73:237-44, 1988) and “Megalign” program(Clewley and Arnold, Methods Mol. Biol, 70:119-29, 1997) can be used toalign sequences and determine similarity, identity, or homology.

In addition to naturally-occurring variations in the MnSOD prohancersequences (e.g., between species), the skilled artisan will furtherappreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, 2, or 5 without altering thefunctional ability of the MnSOD prohancer elements.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules comprising MnSOD prohancer elements that contain changes innucleic acid residues, yet which retain prohancer activity. Mutationscan be introduced into SEQ ID NO:1, 2, or 5 by standard techniques, suchas site-directed mutagenesis and PCR-mediated mutagenesis, and theresultant mutants can be screened for MnSOD prohancer activity toidentify mutants that retain transcriptional regulatory activity.

In addition to the polynucleotides encoding MnSOD prohancer elementsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid sequence. Accordingly, an antisense nucleic acidcan hydrogen bond to a sense nucleic acid.

Given the coding strand or “sense” sequences encoding MnSOD prohancerelements disclosed herein (e.g., SEQ ID NO:1, 2, or 5), antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire MnSOD prohancer element, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe MnSOD prohancer element. An antisense oligonucleotide can be, forexample, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides inlength. An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA comprising an MnSODprohancer element. In one embodiment, an antisense nucleic acid moleculeof the invention can be used to inhibit expression (e.g., basal orinducible expression) of a nucleic acid operatively linked to a MnSODprohancer element, e.g., by forming triple helical structures thatprevent transcription of the target nucleic acids in target cells. Seegenerally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene,C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)Bioassays 14(12):807-15.

The hybridization can be by conventional nucleotide complementarity toform a stable duplex, or, for example, in the case of an antisensenucleic acid molecule which binds to DNA duplexes, through specificinteractions in the major groove of the double helix. An example of aroute of administration of antisense nucleic acid molecules of theinvention include direct injection at a tissue site. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In yet another embodiment, the MnSOD prohancer element nucleic acidmolecules of the present invention can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of MnSOD prohancer element nucleic acid molecules can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication, or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of MnSOD prohancer elements can be modified,(e.g., to enhance their stability or cellular uptake), by attachinglipophilic or other helper groups to PNA, by the formation of PNA-DNAchimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of prohancerelement nucleic acid molecules can be generated which may combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, (e.g., RNAse H and DNA polymerases), to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup B. (1996) supra).The synthesis of PNA-DNA chimeras can be performed as described in HyrupB. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid supportusing standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a between the PNA and the 5′ end of DNA(Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser, K. H. et al. (1975) BioorganicMed. Chem. Lett. 5: 1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

II. Transcriptional Regulation by MnSOD Prohancer Elements

MnSOD prohancer elements of the invention are capable, when induced, ofpromoting transcription of one or more operatively linked heterologouspolynucleotide sequence(s). Accordingly, an important aspect of thepresent invention is the ability to modulate, alter, or regulate, eitherpositively, or negatively, the activity or efficiency of a prohancerelement through the use of transcriptional activators, and particularly,transcriptional inducers.

In particular, prohancer elements of the invention provide for inducibleexpression of sequences that are operatively linked to said elements. Inone embodiment, induction of gene expression is provided by inflammatorymediators. Exemplary inflammatory mediators include for example,lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-α), andinterleukin-1 beta (IL-1β) which are shown in the Examples providedherein to activate, or increase the activity of, the prohancer elements.Other suitable inducers include compounds related to these inflammatorymediators.

For example, inflammatory cytokines can be divided into two groups:those involved in acute inflammation and those responsible for chronicinflammation. IL-1, TNF-alpha, IL-6, IL-11, IL-8 and other chemokines,G-CSF, and GM-CSF play an important role in acute inflammation. Anothersubset of cytokines is involved in chronic inflammation. This lattergroup can be subdivided into cytokines mediating humoral responses suchas IL-4, IL-5, IL-6, IL-7, IL-12 and IL-13, and those mediating cellularresponses such as IL-1, IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, and IL-15,the interferons, and transforming growth factors

In another embodiment, a compound such as 5-amionsalicylic acid can beused to induce a prohancer element of the invention. In fact, the use ofany molecule or inducer which regulates the activity of the disclosedprohancers are contemplated to be useful. The inventors contemplate thatone could either directly contact a transformed cell or animalcomprising the prohancer element constructs disclosed herein with suchan inducer, and thus cause an increase in transcription from theprohancer constructs. Alternatively, contact with one or more substanceswhich increase the production of the inflammatory mediators can be usedto induce a prohancer element of the invention. In the case of an wholeorganism, the activity of a prohancer element could be naturallyactivated in vivo during a physiologic inflammatory response.

In certain embodiments, the heterologous polynucleotide sequence to betranscriptionally controlled (or promoted) by one or more of theprohancer elements include, but are not limited to, heterologousnucleotide sequences encoding heterologous proteins, ribozymes, andantisense constructs.

III. Recombinant Vectors and Host Cells

In another embodiment, the invention provides a vector, preferably anexpression vector, containing one or more MnSOD prohancer elements ofthe invention in opertive linkage with a heterologous polynucleotide(s)of interest, such that expression of the heterologous polynucleotide(s)is under the transcriptional control of the MnSOD prohancer element. TheMnSOD prohancer elements of the invention may be used under theappropriate conditions to direct high level or regulated expression ofthe heterologous polynucleotide(s) either alone, or with a heterologouspromoter. The use of recombinant promoters to achieve protein expressionis generally known to those of skill in the art of molecular biology,for example, see Sambrook et al., (1989). For eukaryotic expression,preferred promoters include those such as a CMV promoter, an RSV LTRpromoter, a β-actin promoter, thymidine kinase promoter, an insulinpromoter, an SV40 promoter.

The MnSOD prohancer elements of the invention may be cloned into anexpression vector in the form of multiple untis, in numerouscombinations and organizations, in forward and reverse orientations, andthe like. The precise optimal location of the prohancer sequences withrespect to the heterologous polynucleotide sequence to be expressed mayvary. A recombinant vector of the invention may also contain otherregulatory elements, e.g., transcription termination sequences,polyadenylation signals, tissue-specific regulatory sequences.

The recombinant expression vectors of the invention can be designed forinducible expression of a protein and/or RNA in prokaryotic oreukaryotic cells. It will be appreciated by those skilled in the artthat the design of a recombinant vector can depend on such factors asthe choice of the host cell to be transformed, the level of expressiondesired, and the like. The recombinant vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, and/or functional RNAs, e.g,antisense RNA or ribozymes, encoded by a polynucleotide operativelylinked to a prohancer element. In certain embodiments, the recombinantvector may be dispersed in a pharmaceutically acceptable solution.

The recombinant vector may be a plasmid, a cosmid, a BAC (bacterialartificial chromosomes), HAC (human artificial chromosomes), YAC (yeastartificial chromosomes), or a viral vector. Viral vectors includereplication defective retroviruses, adenoviruses and adeno-associatedviruses. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ψ Crip, ψCre, ψ2 andψAm. The genome of adenovirus can be manipulated such that it encodesand expresses a heterologous protein or RNA under the control of anprohancer element of the invention, but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See for exampleBerkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991)Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Alternatively, an adeno-associatedvirus vector such as that described in Tratschin et al. (1985) Mol.Cell. Biol. 5:3251-3260 can be used.

Appropriate cloning and expression vectors that may be modified for usewith prohancer elements of the invention, are known in the art, and aredescribed in, for example, Powels et al. (Cloning Vectors: A LaboratoryManual, Elsevier, N.Y., 1985). For other suitable expression systems forboth prokaryotic and eukaryotic cells see chapters 16 and 17 ofSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Another aspect of the invention pertains to host cells containing anucleic acid molecule of the invention, e.g., a prohancer elementoperatively linked to a heterolgous polynucleotide sequence within arecombinant expression vector or a transgene.

Host cells include bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells, mammalian cells, or plantcells. Suitable host cells are discussed further in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Host cells in which the genetic constructs of thepresent invention may be expressed include mammalian cells, and inparticular mammalian cells such as those from a human, monkey, hamster,caprine, feline, canine, equine, porcine, lupine, or murine. A host cellmay also be a cell cultured in vitro or a cell present in vivo (e.g., acell targeted for gene therapy). The host cell can further be afertilized oocyte, embryonic stem cell or any other stem cell used inthe creation of non-human transgenic or homologous recombinant animals.

IV Methods of Nucleic Acid Delivery and DNA Transfection

Nucleic acids of the invention can be introduced into a host cell bystandard techniques for transfecting cells. The term “transfecting” or“transfection” is intended to encompass all conventional techniques forintroducing nucleic acid into host cells, including calcium phosphateco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, and receptor-mediated mechanisms(Curiel et al., 1991; 1992; Wagner et al., 1992a; 1992b). Suitablemethods for transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory textbooks.

The number of host cells transformed with a nucleic acid of theinvention will depend, at least in part, upon the type of recombinantexpression vector used and the type of transfection technique used.Nucleic acids can be introduced into a host cell transiently, or moretypically, for long term regulation of gene expression, the nucleic acidis stably integrated into the genome of the host cell or remains as astable episome in the host cell. Plasmid vectors introduced intomammalian cells are typically integrated into host cell DNA at only alow frequency. In order to identify these integrants, a gene thatcontains a selectable marker (e.g., drug resistance) is generallyintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those which confer resistance tocertain drugs, such as G418 and hygromycin. Selectable markers can beintroduced on a separate plasmid from the nucleic acid of interest or,are introduced on the same plasmid. Host cells transfected with anucleic acid of the invention (e.g., a recombinant expression vector)and a gene for a selectable marker can be identified by selecting forcells using the selectable marker. For example, if the selectable markerencodes a gene conferring neomycin resistance, host cells which havetaken up nucleic acid can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die.

Nucleic acids can also be transferred into cells in vivo, for example byapplication of a delivery mechanism suitable for introduction of nucleicacid into cells in vivo, such as retroviral vectors (see e.g., Ferry, Net al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; and Kay, M. A. etal. (1992) Human Gene Therapy 3:641-647), adenoviral vectors (see e.g.,Rosenfeld, M. A. (1992) Cell 68:143-155; and Herz, J. and Gerard, R. D.(1993) Proc. Natl. Acad. Sci. USA 90:2812-2816), receptor-mediated DNAuptake (see e.g., Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621;Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320), direct injection of DNA (see e.g., Acsadi et al. (1991)Nature 332: 815-818; and Wolff et al. (1990) Science 247:1465-1468) orparticle bombardment (see e.g., Cheng, L. et al. (1993) Proc. Natl.Acad. Sci. USA 90:4455-4459; and Zelenin, A. V. et al. (1993) FEBSLetters 315:29-32). For gene therapy purposes, cells can be modified invitro and administered to a subject or, alternatively, cells can bedirectly modified in vivo.

Liposomes and/or nanocapsules may also be used for the introduction ofpolynucleotides comprising the prohancer elements of the presentinvention into suitable host cells. In particular, the prohancerpolynucleotide compositions of the present invention may be formulatedfor delivery in a solution, such as DMSO, or alternatively encapsulatedin lipid particle, liposomes, vesicle, nanosphere, or nanoparticle.Should specific targeting be desired, methods are available for this tobe accomplished. Antibodies may be used to bind to the liposome surfaceand to direct the antibody and its drug contents to specific antigenicreceptors located on a particular cell-type surface. Carbohydratedeterminants (glycoprotein or glycolipid cell-surface components thatplay a role in cell-cell recognition, interaction and adhesion) may alsobe used as recognition sites as they have potential in directingliposomes to particular cell types. Mostly, it is contemplated thatintravenous injection of liposomal preparations would be used, but otherroutes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the prohancer nucleic acids of the presentinvention. Nanocapsules can generally entrap compounds in a stable andreproducible way (Henry-Michelland et al., 1987). To avoid side effectsdue to intracellular polymeric overloading, such ultrafine particles(sized around 0.1 μm) should be designed using polymers able to bedegraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticlesthat meet these requirements are contemplated for use in the presentinvention, and such particles may be are easily made, as described(Couvreur et al., 1984; 1988).

In certain embodiments, a cell can be transformed in vitro or in vivowith a prohancer element of the invention, either alone or operablylinked to a heterologous polynucleotide sequence (e.g., a transgene),which can be inserted into a particular locus in the cell genome.Methods for gene targeting a site-specific insertion of transgenes intochromosomal DNA are well known in the art and include, for example, themammalian Cre/lox system (Sauer et al. (1998) Methods 14:381-392) orhomologous recombination (see, e.g., U.S. Pat. No. 5,614,396). Bytargeting the prohancer regulatory sequences to locations upstream ofendogenous genes, expression of these genes can be controlledaccordingly. Alternatively, the prohancer elements may regulate theinducible expression of a heterologous transgene.

The present invention is widely applicable to a variety of situationswhere it is desirable to turn gene expression on and off, e.g., toregulate the level of gene expression in a rapid, efficient andcontrolled manner.

For example, the prohancer element can be ligated into a promoterlessexpression vector into which genes of interest can be cloned and usedfor transfection into a variety of cell types. The gene product will beproduced at basal levels unless the prohancer element is activated withLPS, IL-1β, TBF-α, or 5-aminosalicylic acid to stimulate transcription.Thus, gene products can be produced in cells in a timed, coordinatedfashion, and at very high levels. Advantages over other types ofstimulated promoters such as c-fos would be that it is unnecessary toserum starve or to alter the growth media.

Other applications for prohancer elements of the invention include genetherapy. Prohancer elements can be operatively linked to genes ofinterest and introduced into cells either in vivo or in vitro. Theprohancer can then cause inducible expression of the gene upon contactwith non-toxic levels of inflammatory stimuli or 5-aminosalicylic acid.Such stimuli can be introduced into the cell along with theprohancer/gene construct or, during active inflammation, inflammatorystimuli are naturally produced, thereby activating the prohancer toinduce expression of the gene.

Accordingly, in another embodiment, the invention provides an expressionsystem for specifically inducing the expression of a heterologouspolynucleotide. The expression system generally comprises 1) apolynucleotide comprising one or more MnSOD prohancer elements operablylinked to a heterologous polynucleotide of interest, wherein expressionof the heterologous polynucleotide is under the control of the prohancerelement(s), and such that activation of the prohancer results inexpression of the heterologous polynucleotide; and 2) a compound whichactivates the prohancer element, or a polynucleotide encoding a compoundcompound which activates the prohancer element.

The expression system allows for inducible expression of apolynucleotide in a cell. Accordingly, in a further embodiment, theinvention provides a method of achieving inducible transcripton and/orexpression of a heterologous polynucleotide in a cell, comprisingcontacting the cell with an effective amount of a compound whichactivates the prohancer element.

Prohancers of the invention also can be used to cause inducibleexpression of antisense polynucleotides (e.g., ribozymes0 which“down-regulate” the expression of a particular gene or nucleic acid in acell. Ribozymes are catalytic RNA molecules with ribonuclease activitywhich are capable of cleaving a single-stranded nucleic acid, such as anmRNA, to which they have a complementary region. Thus, ribozymes (e.g.,hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature334:585-591)) can be used to catalytically cleave mRNA transcripts tothereby inhibit translation of an unwanted or deleterious mRNA. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in an target mRNA. See, e.g., Cechet al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, known target mRNAs can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

In other aspects of the present invention, the prohancer elements may beused to modulate inducible expression of a heterologous gene. Aheterologous gene may include a native or mutated gene, or a fusiongene. Exemplary heterologous genes which are contemplated to be usefulinclude, but are not limited to, reporter genes (e.g., GFP, GUS, lacZand aequorin), as well as therapeutically beneficial and cytoprotectivegenes.

Genes of particular interest to be expressed in cells of a subject fortreatment of genetic or acquired diseases include those encodingadenosine deaminase, Factor VIII, Factor IX, dystrophin, β-globin, LDLreceptor, CFTR, insulin, erythropoietin, anti-angiogenesis factors,growth hormone, glucocerebrosidase, β-glucouronidase, α1-antitrypsin,phenylalanine hydroxylase, tyrosine hydroxylase, omithinetranscarbamylase, arginosuccinate synthetase, UDP-glucuronysyltransferase, apoA1, TNF, soluble TNF receptor, interleukins (e.g.,IL-2), interferons (e.g., α- or γ-IFN) and other cytokines and growthfactors. Cytoprotective genes or “cytoprotectants” include genes thatprotect a cell against free radical damage, e.g., HO-1, IL-10 and HSP70.

Another embodiment of the invention relates to a method of identifying afactor, e.g., a transcriptional regulatory protein, that interacts witha MnSOD prohancer element, or an inducer of a MnSOD prohancer element.This method generally involves contacting a sample suspected ofcontaining a prohancer-interacting element with a prohancerpolynucleotide composition under conditions effective to allow bindingof the interacting composition to the prohancer element, and detectingthe bound complex. Such methods are particularly desirable indetermining biological components, polypeptides, and peptide fragmentsthat bind to the prohancer compositions of the present invention.

A further aspect of the invention provides methods for screeningcompounds (e.g., peptidemimetics, drugs, small molecules, etc.) whichalter the promoter activity of a MnSOD prohancer element, or alter thebinding of one or more biological components to the prohancer element.The screening for such chemical entities may be performed e.g., by meansof a cell-based assay, an in vitro assay for prohancer function and/orrational drug design. Cell-based assays for screening can be designede.g., by constructing cell lines in which the expression of a reporterprotein, i.e. an easily assayable protein, is dependent on prohanceractivity. Such an assay enables the detection of compounds that alterprohancer activity, either directly or indirectly. Alternatively,compounds that inhibit other cellular functions required for theactivity of one or more gene products placed under the control of such aprohancer element and expressed in a cell in a prohancer-dependentfashion can be identified.

V. Applications/Uses of the Invention

MnSOD prohancer elements of the invention can be used to cause inducibleexpression of a variety of different polypeptides and/or proteins eitherin vivo or in vitro, e.g., in the treatment of diseases. In particular,because MnSOD prohancers are activated by inflammatory stimuli, they canbe used to induce expression of cytoprotective genes during inflammationwhen such inflammatory stimuli are naturally present.

Accordingly, host cells can be engineered, by transformation withprohancer constructs of the present invention, to inducibly turn onexpression of genes encoding selected genes and polypeptides underparticular conditions. Examples of therapeutic proteins which can beexpressed using the prohancer elements include, but are not limited to,CD-4, Factor VIII, Factor IX, von Willebrand Factor, TPA, urokinase,hirudin, interferons, TNF, interleukins, hematopoietic growth factors,antibodies, albumin, leptin, transferrin and nerve growth factors,peptide hormones.

Cells engineered to produce such proteins could be used for either invitro production of the protein or for in vivo, cell-based therapies. Invitro production would entail purification of the expressed protein fromeither the cell pellet for proteins remaining associated with the cellor from the conditioned media from cells secreting the engineeredprotein. In vivo, cell-based therapies would be based on expression ofthe engineered protein.

The cDNA's encoding a number of therapeutically useful human proteinsare available.

VI. Production of Polypeptides In Vitro

Large scale production of a protein of interest can be accomplishedusing cultured cells in vitro which have been modified to contain a geneof interest operatively linked to a prohancer sequence of the invention.For example, mammalian, yeast or bacterial cells can be engineered tocontain these nucleic acid components, as described herein. Themammalian, yeast and bacterial cells can then be cultured using standardtechniques. Expression of the gene and subsequent production of theprotein of interest can then be promoted by treating the cell with aneffective amount of an inducer compound (e.g., 5-aminosalicylic acid,IL-1β, TNF-α, or LPS). such as to activate the prohancer element.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the prohancers of thepresent invention and the gene of interest into the host, availabilityof expression systems, efficiency of expression, stability of the geneof interest in the host, and the presence of auxiliary geneticcapabilities. In general, vectors containing replicon and controlsequences which are compatible with the host cell are used in connectionwith these hosts.

Nucleic acid constructs will include the prohancers of the presentinvention functionally linked to a gene of interest, and optionallyincluding a 3′ end DNA sequence that acts as a signal to terminatetranscription and allow for the poly-adenylation of the resultant mRNA.The vector ordinarily carries a replication site, as well as aselectable marker which is capable of providing phenotypic selection intransformed cells.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e. the untranslated leader sequence, caninfluence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e. to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in animals, and inhumans in particular, will be preferred.

Mammalian cells can be propagated in vitro in two modes: asnon-anchorage dependent cells growing freely in suspension throughoutthe bulk of the culture; or as anchorage-dependent cells requiringattachment to a solid substrate for their propagation (i.e., a monolayertype of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. Large scale suspension culturebased on microbial (bacterial and yeast) fermentation technology hasclear advantages for the manufacturing of mammalian cell products. Theprocesses are relatively simple to operate and straightforward to scaleup. Homogeneous conditions can be provided in the reactor which allowsfor precise monitoring and control of temperature, dissolved oxygen, andpH, and ensure that representative samples of the culture can be taken.

However, suspension cultured cells cannot always be used in theproduction of biologicals. Suspension cultures are still considered tohave tumorigenic potential and thus their use as substrates forproduction put limits on the use of the resulting products in human andveterinary applications (Petricciani, 1987; Larsson and Litwin, 1987).Viruses propagated in suspension cultures as opposed toanchorage-dependent cultures can sometimes cause rapid changes in viralmarkers, leading to reduced immunogenicity (Bahnemann, 1980). Finally,sometimes even recombinant cell lines can secrete considerably higheramounts of products when propagated as anchorage-dependent cultures ascompared with the same cell line in suspension (Nilsson and Mosbach,1987). For these reasons, different types of anchorage-dependent cellsare used extensively in the production of different biological products.

VII. Production of Polypeptides in vivo

Transgenic Organisms

A nucleic acid comprising a prohancer element of the invention, eitheralone or operatively linked to a polynucleotide of interest, can betransferred into a fertilized oocyte of a non-human animal to create atransgenic animal. A transgenic animal is an animal having cells thatcontain a transgene, wherein the transgene was introduced into theanimal or an ancestor of the animal at a prenatal, e.g., an embryonic,stage. A transgene is a DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. In one embodiment, the non-human transgenic animal isa mouse, although the invention is not limited thereto. Transgenicanimals may be useful for the large scale production of proteins, or theproduction of antibodies.

A transgenic animal can be created, for example, by introducing anucleic acid comprising a MnSOD regulatory element operatively linked toa heterologous polynucleotide encoding a gene of interest into the malepronuclei of a fertilized oocyte, e.g., by microinjection, and allowingthe oocyte to develop in a pseudopregnant female foster animal. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene.Methods for generating transgenic animals, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009 and Hogan, B. et al.,(1986) A Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory. A transgenic founder animal can be used to breed additionalanimals carrying the transgene. Transgenic animals carrying a transgeneof the invention can further be bred to other transgenic animalscarrying other transgenes.

It will be appreciated that, in addition to transgenic animals, theregulatory system described herein can be applied to other transgenicorganisms, such as transgenic plants. Transgenic plants can be made byconventional techniques known in the art.

Homologous Recombinant Organisms

The invention also provides a homologous recombinant non-human organismin which a nucleic acid coprising a MnSOD prohancer regulatory elementoperatively linked to a heterologous polynucleotide encoding a gene ofinterest has been introduced into a specific site of the organism'sgenome, i.e., the nucleic acid has homologously recombined with anendogenous gene.

To create such a homologous recombinant organism, a vector is preparedwhich contains DNA encoding the fusion protein flanked at its 5′ and 3′ends by additional nucleic acid of a eukaryotic gene at which homologousrecombination is to occur. Typically, several kilobases of flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see e.g.,Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a descriptionof homologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced DNA has homologously recombined with the endogenous DNAare selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA.

In addition to the homologous recombination approaches described above,enzyme-assisted site-specific integration systems are known in the artand can be applied to the components of the regulatory system of theinvention to integrate a DNA molecule at a predetermined location in asecond target DNA molecule. Examples of such enzyme-assisted integrationsystems include the Cre recombinase-lox target system (e.g., asdescribed in Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res.21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad.Sci. USA 89:7905-7909) and the FLP recombinase-FRT target system (e.g.,as described in Dang, D. T. and Perrimon, N. (1992) Dev. Genet.13:367-375; and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:8469-8473).

VIII. Gene Therapy

MnSOD prohancer elements of the invention have numerous advantageousproperties that render them particularly suitable for application ingene therapy.

Accordingly, an important characteristic of the prohancer elements ofthe invention is that, unlike other inducible regulatory systemsavailable in gene therapy vectors, this sequence contains the capacityto be activated by the body's own response to inflammation through theendogenous release of IL-1β and/or TNF-α. This discovery enablesdevelopment of custom gene therapy vector systems, which are exquisitelycontrolled during an inflammatory response by either elevatingexpression of cytoprotective proteins or, potentially, bydown-regulating deleterious gene products through anti-sensetranscription.

For example, the elements can be used as an “on”/“off” switch for geneexpression that allows for regulated dosing of a gene product in asubject. Indeed, there are several situations in which it may bedesirable to be able to provide a gene product at specific levels and/orat specific times in a regulated manner, rather than simply expressingthe gene product constitutively at a set level.

For example, a gene of interest can be switched “on” at fixed intervals(e.g., daily, alternate days, weekly, etc.) to provide the mosteffective level of a gene product of interest at the most effectivetime. The level of gene product produced in a subject can be monitoredby standard methods (e.g., direct monitoring using an immunologicalassay such as ELISA or RIA or indirectly by monitoring of a laboratoryparameter dependent upon the function of the gene product of interest,e.g., blood glucose levels and the like). This ability to turn “on”expression of a gene at discrete time intervals in a subject while alsoallowing for the gene to be kept “off” at other times avoids the needfor continued administration of a gene product of interest atintermittent intervals. This approach avoids the need for repeatedinjections of a gene product, which may be painful and/or cause sideeffects and would likely require continuous visits to a physician. Incontrast, the system of the invention avoids these drawbacks.

Moreover, the ability to turn “on” expression of a gene at discrete timeintervals in a subject allows for focused treatment of diseases whichinvolve “flare ups” of activity (e.g., many autoimmune diseases) only attimes when treatment is necessary during the acute phase when pain andsymptoms are evident. At times when such diseases are in remission, theexpression system can be kept in the “off” state.

Gene therapy applications that may particularly benefit from the abilityof the prohancer elements of the invention to modulate gene expressionduring discrete time intervals in response to physiologic inflammatorystimuli include both chronic and acute inflammatory conditions such aswound healing, asthma, arthritis, inflammatory bowel disease (e.g.,ulcerative colitis), and cardiovascular disease.

Cells types which can be modified for gene therapy purposes includehematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, skinepithelium and airway epithelium. For further descriptions of celltypes, genes and methods for gene therapy see e.g., Wilson, J. M et al.(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et al.(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et al.(1990) Science 247:1465-1468; Chowdhury, J. R. et al. (1991) Science254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Wilson, J. M. et al. (1992) J. Biol. Chem. 267:963-967;Quantin, B. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Dai,Y. et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; vanBeusechem, V. W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644;Rosenfeld, M. A. et al. (1992) Cell 68:143-155; Kay, M. A. et al. (1992)Human Gene Therapy 3:641-647; Cristiano, R. J. et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126; Hwu, P. et al. (1993) J. Immunol.150:4104-4115; and Herz, J. and Gerard, R. D. (1993) Proc. Natl. Acad.Sci. USA 90:2812-2816.

Gene therapy applications of particular interest in cancer treatmentinclude overexpression of a cytokine gene (e.g., TNF-α) in tumorinfiltrating lymphocytes or ectopic expression of cytokines in tumorcells to induce an anti-tumor immune response at the tumor site),expression of an enzyme in tumor cells which can convert a non-toxicagent into a toxic agent, expression of tumor specific antigens toinduce an anti-tumor immune response, expression of tumor suppressorgenes (e.g., p53 or Rb) in tumor cells, expression of a multidrugresistance gene (e.g., MDR1 and/or MRP) in bone marrow cells to protectthem from the toxicity of chemotherapy.

Gene therapy applications of particular interest in treatment of viraldiseases include expression of trans-dominant negative viraltransactivation proteins, such as trans-dominant negative tat and revmutants for HIV or trans-dominant ICp4 mutants for HSV (see e.g.,Balboni, P. G. et al. (1993) J. Med. Virol. 41:289-295; Liem, S. E. etal. (1993) Hum. Gene Ther. 4:625-634; Malim, M. H. et al. (1992) J. Exp.Med. 176:1197-1201; Daly, T. J. et al. (1993) Biochemistry 32:8945-8954;and Smith, C. A. et al. (1992) Virology 191:581-588), expression oftrans-dominant negative envelope proteins, such as env mutants for HIV(see e.g., Steffy, K. R. et al. (1993) J. Virol. 67:1854-1859),intracellular expression of antibodies, or fragments thereof, directedto viral products (“internal immunization”, see e.g., Marasco, W. A. etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893) and expression ofsoluble viral receptors, such as soluble CD4. Additionally, the systemof the invention can be used to conditionally express a suicide gene incells, thereby allowing for elimination of the cells after they haveserved an intended function.

IX. In Vivo Delivery and Treatment Protocols

As discussed above, in certain applications, it is desirable tointroduce prohancer elements into cells in vivo.

Adenovirus

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express a heterologouspolynucleotide that has been cloned therein.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization or adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5□-tripartite leader (TPL) sequence which makes them preferredmRNAs for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell innoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹, plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

Retrovirus

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5□ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intactsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Other Viral Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Coupar et al., 1988) adeno-associated virus (AAV)(Ridgeway, 1988; Hermonat and Muzycska, 1984) and herpesviruses may beemployed. They offer several attractive features for various mammaliancells (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich etal., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

Non-Viral Vectors

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. As described above, the preferred mechanism for deliveryis via viral infection where the expression construct is encapsidated inan infectious viral particle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA or plasmids. Transfer of the constructmay be performed by any of the methods mentioned above which physicallyor chemically permeabilize the cell membrane. This is particularlyapplicable for transfer in vitro but it may be applied to in vivo use aswell. Dubensky et al. (1984) successfully injected polyomavirus DNA inthe form of calcium phosphate precipitates into liver and spleen ofadult and newborn mice demonstrating active viral replication and acuteinfection. Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of calcium phosphate-precipitated plasmidsresults in expression of the transfected genes. It is envisioned thatDNA encoding a gene of interest may also be transferred in a similarmanner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectarnine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1992a; 1992b). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Eur. Pat. Appl. Publ. No.EP0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type such as lung, epithelialor tumor cells, by any number of receptor-ligand systems with or withoutliposomes. For example, epidermal growth factor (EGF) may be used as thereceptor for mediated delivery of a nucleic acid encoding a gene in manytumor cells that exhibit upregulation of EGF receptor. Mannose can beused to target the mannose receptor on liver cells. Also, antibodies toCD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma)can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues. U.S. Pat. No. 5,399,346(incorporated herein by reference in its entirety) discloses exemplaryex vivo therapeutic methods.

X. Pharmaceutical Compositions

In certain embodiments, it may be desirable to formulate one or morepolynucleotide compositions comprising the prohancer elements of theinvention for administration to a mammal, or even, to a human. As such,it may be important to prepare pharmaceutically-acceptable formulationsof these polynucleotides, or the polypeptides which they produce. Asused herein, the phrase “pharnaceutically-acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or similar untoward reaction when administered to a human, oranimal, as appropriate. The term “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Pharmaceutical compositions may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or they maybe enclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

For oral prophylaxis the polypeptide may be incorporated with excipientsand used in the form of non-ingestible mouthwashes and dentifrices. Amouthwash may be prepared incorporating the active ingredient in therequired amount in an appropriate solvent, such as a sodium boratesolution (Dobell's Solution). Alternatively, the active ingredient maybe incorporated into an antiseptic wash containing sodium borate,glycerin and potassium bicarbonate. The active ingredient may also bedispersed in dentifrices, including: gels, pastes, powders and slurries.The active ingredient may be added in a therapeutically effective amountto a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of an aqueous composition that contains a protein as anactive ingredient is well understood in the art. Typically, suchcompositions are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection can also be prepared. The preparation can alsobe emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

When the route is topical, the form may be a cream, ointment, salve orspray.

The pharmaceutical compositions can be included in a container, pack ordispenser together with instructions for use.

XI. Pharmaceuticals and Methods of Treating Disease

The disclosed compositions may also lend themselves to the developmentof methods for the treatment of various disease states. Treatmentmethods will involve treating an individual with an effective amount ofa viral particle, as described above, containing a therapeutic gene ofinterest. An effective amount is described, generally, as that amountsufficient to detectably and repeatedly to ameliorate, reduce, minimizeor limit the extent of a disease or its symptoms. More rigorousdefinitions may apply, including elimination, eradication or cure ofdisease.

Administration of the therapeutic virus particle to a patient willfollow general protocols for the administration of chemotherapeutics,taking into account the toxicity, if any, of the vector. It isanticipated that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the describedgene therapy.

Where clinical application of a gene therapy is contemplated, it will benecessary to prepare the complex as a pharmaceutical compositionappropriate for the intended application. For example, aqueouscompositions of the present invention comprise an effective amount ofthe compound, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Generally this will entail preparing apharmaceutical composition that is essentially free of pyrogens, as wellas any other impurities that could be harmful to humans or animals. Onealso will generally desire to employ appropriate salts and buffers torender the complex stable and allow for complex uptake by target cells.

A wide variety of disease states may be treated with compositionsaccording to the present invention. In essence, any disease that can betreated by provision of a protein or nucleic acid is amenable to thisapproach. Disease states include a variety of genetic abnormalities suchas diabetes, cancer, cystic fibrosis and various other diseases thatcould be treated by increasing or decreasing expression of a protein ina target cell.

Depending on the particular disease to be treated, administration oftherapeutic compositions according to the present invention will be viaany common route so long as the target tissue is available via thatroute. This includes oral, nasal, buccal, rectal, vaginal or topical.Topical administration would be particularly advantageous for treatmentof skin cancers. Alternatively, administration will be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal or intravenousinjection. Such compositions would normally be administered aspharmaceutically acceptable compositions that include physiologicallyacceptable carriers, buffers or other excipients.

In certain embodiments, ex vivo therapies also are contemplated. Ex vivotherapies involve the removal, from a patient, of target cells. Thecells are treated outside the patient's body and then returned. Oneexample of ex vivo therapy would involve a variation of autologous bonemarrow transplant. Many times, ABMT fails because some cancer cells arepresent in the withdrawn bone marrow, and return of the bone marrow tothe treated patient results in repopulation of the patient with cancercells. In one embodiment, however, the withdrawn bone marrow cells couldbe treated while outside the patient with an viral particle that targetsand kills the cancer cell. Once the bone marrow cells are “purged,” theycan be reintroduced into the patient.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses in association with itsadministration, i.e., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime. Unit dose of the present invention may conveniently may bedescribed in terms of plaque forming units (pfu) of the viral construct.Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³ pfu and higher.

Preferably, patients will have adequate bone marrow function (defined asa peripheral absolute granulocyte count of >2,000/mm³ and a plateletcount of 100,000/mm³), adequate liver function (bilirubin ≦1.5 mg/dl)and adequate renal function (creatinine <1.5 mg/dl).

Viral vectors may be employed to deliver therapeutic genes to cancercells. Target cancer cells include cancers of the lung, brain, prostate,kidney, liver, ovary, breast, skin, stomach, esophagus, head & neck,testicles, colon, cervix, lymphatic system and blood. Of particularinterest are non-small cell lung carcinomas including squamous cellcarcinomas, adenocarcinomas and large cell undifferentiated carcinomas.

According to the present invention, one may treat the cancer by directlyinjecting a tumor with the viral vector. Alternatively, the tumor may beinfused or perfused with the vector using any suitable delivery vehicle.Local or regional administration, with respect to the tumor, also iscontemplated. Finally, systemic administration may be performed.Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours,to about 12-24 hours, to about 1-2 days, to about 1-2 weeks or longerfollowing the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs.

For tumors of ≧4 cm, the volume to be administered will be about 4-10 ml(preferably 10 ml), while for tumors of <4 cm, a volume of about 1-3 mlwill be used (preferably 3 ml). Multiple injections delivered as singledose comprise about 0.1 to about 0.5 ml volumes. The viral particles mayadvantageously be contacted by administering multiple injections to thetumor, spaced at approximately 1 cm intervals.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic viral constructsmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional viraltreatments subsequent to resection will serve to eliminate microscopicresidual disease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, will involve multiple doses. Typical primary tumor treatmentinvolves a 6 dose application over a two week period. The two weekregimen may be repeated one, two, three, four, five, six or more times.During a course of treatment, the need to complete the planned dosingsmay be reevaluated.

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil,bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxol,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate.

Combination radiation therapies may be x- and -irradiation. Dosageranges for x-irradiation range from daily doses of 2000 to 6000roentgens for prolonged periods of time (3 to 4 weeks), to single dosesof 2000 to 6000 roentgens. Dosages for radioisotopes vary widely, anddepend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by neoplastic cells.

Various combinations may be employed, gene therapy is “A” and the radio-or chemotherapeutic agent is “B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/AA/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/AB/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to well known parameters.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES

Experimental Procedures

Cell Culture and Transfection

L2 cells, a rat pulmonary epithelial-like cell line (ATCC CCL149), weregrown in Ham's modified F12K medium (GIBCO) with 10% fetal bovine serum(Flow Laboratories), ABAM (Penicillin G 100 U/ml, Streptomycin 0.1mg/ml, Amphotericin B 0.25 μg/ml (Sigma) and 4 mM glutamine at 37° C. inroom air, 5% CO₂. Transfections were carried out using a batchtransfection method. Cells were grown on 150 mm tissue culture platesuntil 70-90% confluent. The cells were transfected with 10 μg of eachexpression vector using a modified DEAE-dextran method (Kriegler, 1990).After 24 hours, cells from each 150 mm, batch transfected plate weretrypsinized, pooled, and plated onto four separate 100 mm tissue cultureplates. After 24 hours, inflammatory mediators were added to the mediumof each plate with final concentrations of 0.5 μg/ml LPS, 10 ng/mlTNF-α, or 2 ng/ml IL-1β. Twenty four hours later, total RNA was isolatedfrom the cell monolayers for northern analysis. For the human growthhormone protein assay, individual 100 mm plates were allowed to incubatefor 48 hours after the addition of LPS, TNF-α or IL-1β before mediasamples were collected. Each vector was tested in n≧4 independentexperiments to evaluate the reproducibility and effectiveness of eachconstruct.

Generation of Reporter Constructs

From the 17 kb rat MnSOD genomic clone, a 4.5 kb EcoRI/EagI fragment of5′ untranslated sequence was isolated and the 5′ overhang ends filled inusing the Kienow fragment of E. coli DNA polymerase I. The resultingblunt-end EcoRI/EagI fragment was cloned into the HincII polylinker sitein a promoterless, pUC 12-based human growth hormone expression vector,pØGH, (Selden et al., 1986), creating a 9.3 kb plasmid referred to asEco/E GH. Unique restriction enzyme sites were utilized to deleteincreasing portions of the MnSOD Eco/E sequence, creating the promoterdeletion vectors illustrated in FIG. 1A. To test for non-specificeffects of the inflammatory mediators on hGH expression, we used an hGHexpression vector, pTKGH, that contains a minimal promoter from herpessimplex thymidine kinase (Selden et al., 1986).

To identify possible regulatory elements in the MnSOD gene that mightinteract with the 5′ promoter and induce and/or enhance expression oftransgene sequences, an internal 6.1 kb HindIII fragment (+1180 to+7312) from the rat MnSOD genomic clone was cloned into the HindIII siteof pØGH vector containing the Hind/E promoter fragment, creating a 13.45kb vector (FIG. 2A). The same 6.1 kb HindIII fragment was also clonedinto the pØGH vector containing the Hind/E promoter fragment in theopposite orientation (e.g., 3′→5′).

To localize enhancer activity within the 6.1 kb HindIII fragment, the3.8 Kb HindIII/HpaI fragment (+1180 to +5046) and the 2.3 KbHpaI/HindIII fragment (+5046 to +7312) (both part of the 6.1 kbfragment) were independently cloned in both orientations into the pØGHvector containing the Hind/E promoter fragment. To clarify the positionof the enhancer within the 3.8 Kb HindIII/HpaI fragment, serial 3′ and5′ deletions of this fragment were generated (FIGS. 3A and 4A) andcloned into the pØGH vector containing the Hind/E promoter fragment atthe HindIII site or the NdeI site. Oligonucleotides flanking the regionsof interest were designed containing either Hind III or NdeI sites forconvenient ligation into the restriction sites.

To test whether the enhancer activity was specific to the MnSODpromoter, the plasmid pTKGH, which contains a heterologous,TATA-containing, non-GC-rich promoter, was also used. The 6.1 kb HindIIIMnSOD genomic fragment (+1180 to +7312) was cloned into the HindIII sitein the pTKGH polylinker in both orientations (FIG. 6A). To evaluate theinteraction of the MnSOD promoter with the internal enhancer fragment,the 919 bp fragment of the MnSOD gene containing the entire enhancerregion was ligated into the NdeI site of the promoter deletionconstructs previously described (see FIGS. 3A and 8A).

Comparison of the rat MnSOD enhancer sequence with the analogous regionin intron 2 of the human MnSOD gene revealed a high degree of homologyin intron 2 (FIG. 5). PCR was used to amplify a 466 bp fragment (+2410to +2875, human MnSOD, GenBank Accession No. S77127) from human genomicDNA using the following primers: 5′-CGTTAGTGGTTTGCACAAGGAAGATAATCG-3′(SEQ ID NO:3) 5′-GGCTCTGATTCCACAAGTAAAGGACTG-3′ (SEQ ID NO:4)The human MnSOD enhancer fragment was inserted into the pTKGH vector inboth orientations (FIG. 6A). The 466 bp human enhancer fragment and the260 bp, 553 bp or 746 bp rat enhancer fragments were also ligated intothe pØGH vector (see FIG. 9A).RNA Isolation and Northern Analysis

Inducible gene expression in transfected cells was analyzed by directlymeasuring hGH mRNA and/or endogenous MnSOD mRNA levels. Total RNA wasisolated by the guanidinium thiocyanate-phenol-chloroform extractionmethod described by Chomczynski and Sacchi (1987) with modifications.Twenty μg of RNA was size fractionated on a 1% denaturing agarose gel,transferred to a nylon membrane and UV-crosslinked. Membranes werehybridized with ³²P-labelled cDNA probes from the appropriate genes(e.g., hGH, MnSOD) generated by random primer extension. Membranes werereprobed with cathepsin B as a loading control.

Human Growth Hormone Assay

The concentration of secreted hGH was measured using an¹²⁵Iodine-labeled monoclonal antibody assay kit purchased from NicholsInstitute with a lower limit sensitivity of 0.06 ng/ml. Eachexperimental sample was assayed in duplicate.

Human growth hormone assay data from similarly transfected and treatedplates from all experiments were combined. To determine whethertreatment with inflammatory mediators increased expression of the hGHreporter, the mean concentration of hGH from all plates transfected withthe same vector and treated with the same agonist was compared tountreated transfected control plates (n≧16). We also tested fordifferences in basal and stimulated hGH expression between vectors. Astudent's unpaired, two-tailed t test or two way analysis of variancewas performed, and a p value of <0.05 was considered significant.

Electrophoretic Mobility Shift Assays (EMSA)

EMSAs were performed as previously described (Fried and Crothers, 1981)with 8 μg nuclear extract prepared from control and LPS, TNF-α, or IL-1βtreated L2 cells by high salt extraction (Andrews and Faller, 1991).Binding reactions were carried out at room temperature in 10 mM HEPES,pH 7.9, 100 mM KCl, 1 mM dithiothreitol (DTT), 0.5 mM MgCl₂, 0.1 mM EDTAand 8.5% glycerol, to yield a final volume of 20 μl. EMSA probes weremade from cloned PCR products of the MnSOD enhancer region between +4130and +4491 of the rat MnSOD gene. Probes were end-labeled by filling therecessed 3′ termini of EcoRI digested fragments with ³²P-dATP using theKlenow fragment of E. coli DNA polymerase I. Fragments used in EMSAswere 3′ 143 bp (+4348 to +4491), 5′ 143 bp (+4231 to +4374), 100 bp(+4231 to +4331), 95 bp (+4331 to +4426), and 103 bp (+4426 to +4529).Numbers in parentheses refer to the sequence of the rat MnSOD gene,GenBank Accession No. X56600.

Example 1 Characterization of the Rat MnSOD Promoter

The following studies were performed to characterize the region of therat MnSOD gene responsible for induction of gene expression in responseto inflammatory mediators.

The Rat MnSOD Promoter does not Contain all the DNA Elements Necessaryfor Cytokine-Inducible Expression

MnSOD gene expression is stimulated by inflammatory mediators, includingLPS, TNF-α, and IL-1β, and is due at least in part to de novo genetranscription. To identify sequences in rat MnSOD which conferspecificity for LPS-, TNF-α- and IL-1β-dependent gene induction, aseries of MnSOD promoter deletion constructs were generated using thepromoterless human growth hormone vector pØGH (FIG. 1A). Uniquerestriction sites within the rat MnSOD 5′ flanking sequence were used tocreate vectors containing the promoter deletions. Each 5′ promoterdeletion construct contained sequences which incorporated the MnSODtranscriptional start site. Cells transfected with a pØGH reportervector containing a 4.5 Kb fragment (Eco/E) of the rat MnSOD promotershowed a 2 to 3 fold induction of hGH mRNA levels in response tostimulation with LPS, TNF-α, and IL-1β (FIGS. 1B and 1C). To assure thathGH in the medium reflected the majority of total hGH protein producedby the transfected cells, hGH was also measured in the cell monolayer.Only 2-3% of total hGH was retained in the cells and 97% was secretedinto the medium. As the MnSOD promoter was progressively shortened, thedeletions had no effect on either basal or stimulated hGH expression(mRNA or protein), until the MnSOD promoter fragment was shortened fromthe SacII restriction site to the NaeI site at which point allexpression was lost. Messenger RNA (FIG. 1B) and protein levels (FIG.1C) of hGH were comparable suggesting that cytokines did notsignificantly affect translation, post-translational modification orsecretion.

The promoter deletion data suggests that the 5′ flanking sequence of therat MnSOD gene between positions −154 and +32 contains cis-actingelements necessary for basal and a small amount of inducible MnSODexpression. Relative to the ten protein binding sites identified in theMnSOD promoter by in vivo footprinting experiments, these data wouldindicate that the five sites most proximal to the transcriptionalinitiation site (within the Sac II fragment) are responsible for the 2to 3 fold increase in hGH levels compared to controls. However, themagnitude of induction of hGH mRNA and protein levels in the pØGH MnSODpromoter deletion constructs is low (only a 2 to 3 fold induction)compared to the induction of endogenous MnSOD as evaluated by northernanalysis. In addition, these constructs contained only 5′ flankingsequences of the rat MnSOD gene, which incorporated only one of theseven DNaseI hypersensitive sites identified within the MnSOD gene (seeFIG. 2A). Therefore, other regulatory elements outside of the 5′proximal promoter must be involved in the induction of MnSOD geneexpression by LPS, TNF-α and IL-1β.

Example 2 Characterization of MnSOD Enhancer Elements

The studies described in Example 1 showed that the rat MnSOD gene 5′proximal promoter was not solely responsible for inducible geneexpression. Accordingly, the following studies were performed toidentify and characterize further transcriptional regulatory elementswithin the MnSOD gene itself (e.g., within intron sequences).

A Novel Inducible Cis-Acting Enhancer Element Exists within the Rat andHuman MnSOD Genes

To determine whether the remaining DNaseI hypersensitive sites describedin Example 1 (within the MnSOD gene) contained regulatory function, pØGHexpression vectors were created that contained both a 2.5 Kb fragment ofthe MnSOD promoter (Hind/E) and a 6.1 kb HindIII internal fragment ofthe MnSOD gene which contains all of the other Dnase I hypersensitivesites (FIG. 2A). Expression of hGH mRNA in cells transfected with thisvector was compared to expression of hGH in cells transfected with apØGH construct containing only the 5′ Hind/E promoter fragment.Inclusion of the 6.1 Kb HindIII internal MnSOD genomic fragment,resulted in a robust induction (10-15 fold) of hGH mRNA levels inresponse to LPS, TNF-α and IL-1β stimulation, and the effect wasindependent of the orientation of the 6.1 kb HindIII fragment within thevector (FIG. 2B).

Furthermore, the 6.1 Kb Hind III internal MnSOD fragment was cleaved atthe unique HpaI site, and the enhancer activity of the resulting 3.8 Kband 2.3 Kb internal MnSOD fragments were tested using the pØGH vector.The cis-acting element responsible for the inducible activity waslocalized to the 3.8 Kb fragment, which represents the 5′ half of theoriginal 6.1 Kb HindIII fragment (FIG. 2C). Again, orientation did notinfluence the inducible activity in response to inflammatory mediators,thus indicating that this element has the properties of an enhancer.

Localization of the Novel MnSOD Inducible Enhancer Element within Intron2

To further localize the region of the inducible cis-acting elementwithin the 3.8 kb HindIII-HpaI MnSOD fragment, the polymerase chainreaction (PCR) was used to amplify serial deletion fragments (from boththe 5′ and 3′ ends) spanning this region (FIG. 3A). Oligonucleotidescomplementary to regions of the 3.8 kb HindIII-HpaI fragment weregenerated containing restriction sites (HindIII or NdeI) to facilitatecloning of the enhancer fragments into the pØGH vector containing theHind/E fragment of the MnSOD promoter. The inducible enhancer activitywas assessed by transient transfection of the pØGH vector constructsinto L2 cells and northern analysis.

Multiple 3′ and 5′ deletions of the 3.8 kb HindIII-HpaI enhancerfragment demonstrated inducible activity that was comparable to theinduction of endogenous MnSOD mRNA levels. Based on the 3′ deletions ofthis 3.8 Kb internal MnSOD fragment, the inducible enhancer activity waslocalized to the 464 bp-1.0 Kb fragment in the 5′ end of the fragment(FIGS. 3B and 3C). This position coincides to DNase hypersensitive site2 close to the intron 2—exon 3 boundary. 5′ deletion analysis of the 3.8Kb internal MnSOD fragment indicated that the 338 bp and 227 bpfragments show reduced enhancer activity relative to the 455 bp fragment(FIGS. 3D and 3E). Taken together, these data indicate that the MnSODenhancer activity regulated by LPS, TNF-α, and IL-1β appears to existwithin a 200 to 300 bp region near the 3′ end of intron 2.

The Enhancer is Composed of a Complex Set of Interacting Elements

To further characterize the enhancer within the 3′ region of intron 2 ofthe rat MnSOD gene, additional deletion constructs were created whichcontained either 260 bp or 143 bp fragments of the intron 2 region. PCRamplification was used to generate a 260 bp fragment spanning the regionbetween the two 5′ deletions (455 bp and 227 bp) shown in FIG. 3A, aswell as two 143 bp fragments which overlap each other within this 260 bpfragment (FIG. 4A). This region was felt to contain the entireregulatory sequence responsive to LPS, TNF-α, and IL-1β. The ability ofthese fragments to cause inducible expression was evaluated by transienttransfection and northern analysis. The 260 bp fragment contained allthe enhancer activity seen in the larger 919 bp fragment (FIG. 4B), andretained responsiveness to all of the inflammatory mediators (e.g., LPS,TNF-α, and IL-1β). The two 143 bp fragments retain enhancer activity,although possibly less than the full 260 bp region (FIG. 4C). Theseresults are consistent with the deletion analysis, which showed partialenhancer activity in the 338 bp fragment compared to the 455 bpfragment, and a total loss of activity with the 227 bp fragment (FIG.3E). This is also consistent with the fact that the 260 bp fragmentspans the region between the 455 bp deletion fragment and the 227 bpdeletion fragment (FIG. 3A). Taken together, the results summarized inFIGS. 3E and 4C demonstrate that the 260 bp fragment delineates theminimum functional boundaries of the MnSOD enhancer.

To evaluate protein binding to the MnSOD enhancer elements,electrophoretic mobility shift assays were performed with nuclearextracts from control and treated (LPS, TNF-α, IL-1β) cells. FIG. 4Dshows the difference in the protein binding patterns between treated andcontrol nuclear extracts as well as the different patterns of proteinbinding between the two functional 143 bp DNA fragments that comprisethe enhancer element. Constitutive protein binding is observed in the 3′143 bp fragment, but several inducible DNA-protein complexes can beappreciated in both fragments. In an attempt to further localize theprotein binding sites, smaller fragments of the enhancer region weregenerated by PCR and evaluated by electrophoretic mobility shift assays(FIG. 4E). Once again, constitutive protein binding was observed in the100 bp and 103 bp fragments. However, as with the 143 bp fragments,inducible protein binding was also observed in two of the smallerfragments (100 bp and 95 bp), most notably in the 95 bp fragment whichshows a dramatic difference in binding between control and treatednuclear extracts. Further localization of protein binding was attemptedwith 50 bp and 30 bp fragments from the enhancer region, however,stimulus specific protein-DNA complexes were not observed. Therefore,protein-protein interactions are most likely a prerequisite for specificDNA binding, thus explaining the loss of stimulus-specific protein-DNAinteractions in the smaller deletions of this complex regulatoryelement.

The Rat and Human MnSOD Gene Enhancers Act with a Heterologous Promoter

Sequence analysis identified a high level of homology between thesequence of intron 2 of the rat MnSOD gene, comprising the enhancerelement, and the corresponding region in the human MnSOD gene (FIG. 5).

To determine whether the MnSOD enhancers could function with aheterologous promoter, rat and human enhancer fragments were cloned intothe pTKGH expression vector (FIG. 6A). The herpes virus thymidine kinasepromoter in this vector is a 200 bp minimal, TATA containing promoter,quite dissimilar unlike the GC-rich, TATA- and CAAT-less MnSOD promoter.Results of transient transfections and northern analysis of the 6.1 kbHindIII internal MnSOD fragment in pØGH showed that the rat cis-actingenhancer element dramatically increased transcriptional activity inresponse to inflammatory mediators (FIG. 6B). Cells treated with LPS,TNF-α, and IL-1β had marked levels of hGH mRNA in comparison with almostundetectable levels in control, untreated cells. The rat MnSOD enhancerelement functioned equally well in both orientations. The ability ofthis novel element to enhance transcriptional activity of a heterologouspromoter in an orientation-independent and position-independent mannerfurther qualifies it as an enhancer element.

In order to evaluate the functional significance of the homologous humanMnSOD enhancer region, a 553 bp fragment of the rat enhancer and ananalogous 466 bp region (generated by PCR amplification from humangenomic DNA) from intron 2 of the human MnSOD gene were inserted intothe pTKGH vector. The ability of the fragments to cause inducibleenhancer activity was assessed by transient transfection and northernanalysis. Both the rat and the human MnSOD enhancer fragments promotedessentially identical inducible gene expression in response to LPS,TNF-α and IL-1β (FIG. 6C), indicating that the analogous region ofintron 2 of the human MnSOD gene likely acts as an enhancer in theendogenous gene, and that the enhancer element itself is well conservedbetween species.

Rat intestinal epithelial cells were also transfected with a pTKGHvector containing a 508 bp fragment of the MnSOD enhancer element andtreated with both mesalamine (5-ASA, 2 mg/ml) and LPS (0.5 μg/ml) for 8hours. Total RNA was isolated and rat MnSOD and hGH mRNA levels wereevaluated by northern analysis (FIG. 7). Both endogenous MnSOD and thehuman growth hormone reporter gene were induced by mesalamine and LPS inan identical fashion, indicating that the enhancer element is responsiveto both endogenous inflammatory stimuli as wells exogenouspharmaceutical compositions.

Characterization of MnSOD Promoter-Enhancer Interactions

In vivo footprinting has demonstrated that the MnSOD 5′ proximalpromoter (described in Example 1) contains 10 potential protein bindingsites. These sites are illustrated in FIG. 8A relative to therestriction sites that were employed for the promoter deletion analysis((●), FIG. 1A). To define the areas within the MnSOD promoter requiredfor interaction with the enhancer element, the rat MnSOD promoterdeletion constructs were coupled with a 919 bp enhancer fragment in thepØGH vector (FIG. 8A). Transient transfection and northern analysis wereused to evaluate the inducible activity of these constructs. The fivemost distal protein binding sites in the MnSOD 5′ proximal promotercould be deleted without any detectable decrease in inducible activity(FIGS. 8B and 8C). However, when the remaining five proximal bindingsites in the MnSOD 5′ proximal promoter were eliminated, almost completebasal activity was lost (FIG. 8C). LPS-inducible transcription couldstill occur when all of the protein binding sites in the MnSOD promoterwere deleted (Nae/E deletion construct), but only when the vectorconstruct contained the 919 bp enhancer fragment (FIG. 8C). In theabsence of the enhancer element, deletion of the same promoter regioneliminated any transcription (FIGS. 1B and 1C). These studiesdemonstrate that a minimal MnSOD promoter deletion fragment (Nae/E) isnot capable of supporting significant levels of transcription by itself,but can support inducible transcription when coupled to the MnSODenhancer.

Characterization of Rat and Human MnSOD Enhancer Elements asAdditionally having Promoter Activity

To determine whether the MnSOD enhancer element might exhibitstimulus-dependent promoter activity in the absence of a true promoter,the rat and the human enhancer elements were inserted into thepromoterless pØGH vector and the pTKGH vector (FIGS. 9A and 9B). FIG. 9Ashows the results of the 466 bp human MnSOD enhancer fragment in thepresence (pØGH ) and absence (pTKGH) of the TK promoter. The MnSODenhancer alone mediated inducible transcription in response to LPS,TNF-α and IL-1β stimulation, but at lower levels than when the enhancerwas coupled with the TK promoter. Interestingly, two transcripts, onethe correct size and one larger, were seen when the human 466 bpenhancer fragment acted as its own promoter. When the 553 bp and 746 bprat enhancer fragments were tested in the pØGH vector, similar resultsto the human enhancer element were obtained, in that, twodifferent-sized transcripts were observed (FIG. 9B). Of note, however,is that when the 553 bp fragment containing the enhancer was inserted infront of the hGH gene in the 3′→5′ orientation, the correct-sizedtranscript predominated. Furthermore, as can be seen in FIG. 7B, whenthe rat 260 bp enhancer fragment acting as a promoter was inserted intothe pØGH vector in the 3′→5′ orientation, a single stimulus-responsivetranscript of the correct size resulted.

These studies indicate that the MnSOD enhancer can indeed actindependently as a promoter, and that orientation and position relativeto the start of transcription were important for promoter function. Thefinding that the MnSOD enhancer has both inducible enhancer and promoteractivity is also referred to herein as the identification of a novelprohancer element.

Discussion

Extensive studies have been performed on the molecular regulation of theMnSOD gene in a variety of mammalian cells including: pulmonaryendothelial (Visner et al., 1992) and epithelial cells (Visner et al.,1990); intestinal epithelial (Valentine and Nick, 1992), smooth muscle(Tannahill et al., 1997) and myenteric neurons (Valentine et al., 1996);mesangial (Stephanz et al., 1996) and glomerular epithelial cells(Gwinner et al., 1995); neurons and glial cells (Kifle et al., 1996); aswell as primary hepatocytes cultures (Dougall and Nick, 1991).

In many cells, MnSOD levels are dramatically induced by LPS, IL-1β, andTNF-α with induction levels ranging from 15-100 fold depending on thecell type (FIG. 12). One exception is that hepatocytes show responsesprimarily with IL-6 and INF-γ. The extension of these results has led tothe identification of an extremely potent enhancer element, describedherein, which mediates the regulation of the MnSOD gene in an LPS-,IL-1β-, and TNF-α-dependent manner. This element can function in anorientation and position independent manner, and also retains theability to function as a totally independent stimulus-responsivepromoter. In addition, this element has been evolutionarily conservedbetween rodents and man.

Another characteristic of this enhancer is its ability to function witha TATA- and CAAT-box containing minimal promoter, whereas the endogenousMnSOD promoter lacks these elements. The potency of the enhancer isincreased with a minimal viral thymidine kinase promoter, exhibitingstimulus-specific induction levels of up to 45 fold. Given theinducibility, minimal size, and stimulus-specificity of this enhancer,it appears to be an ideal regulatory element for gene therapy. Theinherent ability of this element to respond to IL-1β and TNF-α willallow this enhancer to drive transgene expression, when and only whenendogenous levels of IL-1β and TNF-α are increased. Therefore, thetransgene expression will directly mimic the changes in cellular IL-1βand TNF-α levels, which occur during acute and chronic inflammation.Moreover, this enhancer element will also respond during inflammatorysituations associated with bacterial infections where systemic LPSconcentrations presumably increase, analogous to Pseudomonas aeruginosacolonization in cystic fibrosis patients.

Example 3 Construction of Recombinant Adeno-Associated Virus (AAV)Vectors Containing MnSOD Prohancer Elements

The novel MnSOD prohancer elements of the invention can also be used togenerate delivery vectors suitable for inducible heterologous geneexpression. A series of AAV vectors can be engineered containing theMnSOD prohancer element. To do this, the P_(CMV) promoter is removedfrom pTR-UF2 using KpnI and XhaI, and the MnSOD prohancer, either aloneor coupled to a minimal promoter, is inserted (FIG. 10). The recombinantAAV plasmids are packaged using the current method for isolating rAAV asdeveloped by Hermonat and Muzyczka (1984). Human cells (e.g., 293 cells)are transfected with the plasmid which consists of a transgene flankedby the AAV terminal repeats (TR), the only AAV sequences required forviral DNA replication, packaging and integration. The cells are alsotransfected with a complementing plasmid that is defective forpackaging, but supplies the wild type AAV rep and cap genes in trans.Finally, the cells are infected with adenovirus to supply the adenovirushelper functions, E1A, E1B, E2A, E4, and VA. The rAAV virus stockproduced contains both adenovirus and the AAV recombinant virus.

A fast and reproducible protocol has developed for the purification andconcentration of rAAV. This protocol is based on partial purification ofthe initial freeze/thaw lysate by ammonium sulfate fractionation,followed by ion exchange batch chromatography and a final CsCl gradientcentrifugation step. The helper Ad virus is eliminated byheat-inactivation early in the process and is no longer present as adistinct band in the CsCl gradient. A PCR based assay is employed tomonitor rAAV-positive fractions from the CsCl gradient. The final rAAVstock is titered by the infectious center assay and a QC-PCR assay. Byoptimizing the transfection protocol, the time of addition ofadenovirus, the pH of the extraction buffer, and the method ofpurification, a typical yield is about 30-50 infectious units per cell.A preparative isolation from a total of twenty 15 cm tissue culturedishes provides a final yield of approximately 10¹⁰ infectious units ofrAAV in a total volume of 0.5 ml. Quality control assays for purity andinfectivity are performed, as appropriate.

Packaged vectors are used to transduce cell cultures (e.g., A549 cellsand astrocyte cultures) at a multiplicity of 1, 10 and 100 infectiousunits per cell. Cells are selected with G418 at 2 to 3 days aftertransduction. Selection is continued for 10 days followed by isolationof individual clones using cloning cylinders. Green fluorescent protein(GFP) will be assayed using phase contrast and epifluorscence microscopyfollowed by northern analysis to detect the GFP message. The AAVcassette in vector pTR-UF3 may also be used (FIG. 10), which allows fora dicistronic expression system in which the P_(CMV) promoter is removedand the MnSOD prohancer element inserted, and a transgene of interest isinserted via the multiple cloning sites in the flanking polio IRESelement.

AAV vectors are employed for gene targeting of candidate genes (e.g.,CFTR) that are ideally suited to test the unique characteristics of the“prohancer” (FIG. 11). One of the difficulties associated with the useof AAV in cystic fibrosis is the packaging limit of the virion (˜5 kb),based on the large size of the CFTR coding sequence. To address thisissue, a CFTR minigene has been generated with a 118 amino acid deletionwhich retains 75% of the channel activity as confirmed by using ³⁶C⁻isotope tracer efflux. The 260 bp MnSOD enhancer element may be insertedin the unique BgIII site 5′ to the minimal TK promoter alreadyincorporated in this vector. An alternative construct involves theligation of the prohancer into a unique XhoI site 5′ to the full lengthCFTR coding region. It is possible that with the incorporation of the260 bp prohancer fragment that this rAAV vector (5.09 kb) will be largerthan the packaging limit of the AAV virion. Given this possibility,exonuclease digestion of the XhoI digested vector may be used toeliminate 120-145 bp of vector sequence between the left ITR and thebeginning of the CFTR coding sequence, followed by a blunt-end ligationof the prohancer element. These constructs are packaged, produced on alarge scale and subsequently transduced into cells, as described above.

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1. An isolated manganese superoxide dismutase regulatory elementconsisting of the nucleotide sequence of SEQ ID NO:1 or SEQ NO:5, theregulatory element being capable of causing inducible transcription orexpression of an operably linked heterologous polynucleotide.
 2. Theisolated regulatory element of claim 1 operably linked to a heterologouspolynucleotide so that, upon activation of the regulatory element,transcription or expression of the heterologous polynucleotide isinduced.
 3. An isolated polynucleotide comprising a manganese superoxidedismutase regulatory element operably linked to a heterologouspolynucleotide, wherein the regulatory element comprises the nucleotidesequence of SEQ ID NO:5, the regulatory element being capable of causinginducible transcription or expression of the operably linkedheterologous polynucleotide.
 4. The isolatead polynucleotide of claim 3,wherein the regulatory element comprises the nucleotide sequence of SEQID NO:1.
 5. The isolated polynucleotide of claim 3, further comprising aheterologous polynucleotide operably linked to the regulatory element sothat, upon activation of the regulatory element, transcription orexpression of the heterologous polynucleotide is induced.
 6. Theisolated polynucleotide of claim 1, wherein the heterologouspolynucleotide encodes a cytoprotectant.
 7. The isolated polynucleotideof claim 1 which induces transcription or expression of an operativelylinked heterologous polynucleotide in the presence of an inflammatorystimulus.
 8. The isolated polynucleotide of claim 9, wherein theinflammatory stimulus is selected from the group consisting of TNF-α,IL-1β, and LPS.
 9. The isolated polynucleotide of claim 1 which inducestranscription or expression of an operatively linked heterologouspolynucleotide in the presence of 5-aminosalicylic acid.
 10. Theisolated polynucleotide of claim 1, wherein the regulatory sequence isoperatively linked to a promoter sequence.
 11. The isolatedpolynucleotide of claim 10, wherein the promoter is the Herpes simplexthymidine kinase promoter.
 12. A cell transformed with the isolatedpolynucleotide of claim
 1. 13. An inducible expression systemcomprising: a) an isolated polynucleotide comprising a regulatoryelement, wherein the regulatory element comprises a nucleotide sequenceselected from the group consisting of SEQ ID NO:5, SEQ NO:1 andnucleotide sequences having at least about 90% identity to SEQ ID NO:1,wherein the regulatory element induces transcription or expression of anoperably linked heterologous polynucleotide upon activation; and b) acompound which activates the regulatory element, or a polynucleotideencoding a compound which activates the regulatory element.
 14. Theexpression system of claim 13 wherein the regulatory element is a humanregulatory element
 15. The expression system of claim 13 wherein theregulatory element is a rat regulatory element
 16. The expression systemof claim 13 wherein the compound which activates the regulatory elementis an inflammatory stimulus.
 17. The expression system of claim 16wherein the compound which activates the regulatory element is selectedfrom the group consisting of TNF-α, IL-1β, and LPS.
 18. The expressionsystem of claim 13 wherein the compound which activates the regulatoryelement is 5-aminosalicylic acid.
 19. The expression system of claim 13further comprising a heterologous polynucleotide operably linked to theregulatory element.
 20. The expression system of claim 13 furthercomprising a promoter operably linked to the regulatory element.
 21. Amethod of producing a polypeptide comprising introducing the expressionsystem of claim 19 into a cell under conditions suitable for expressionof the heterologous polypeptide.
 22. A method of achieving inducibletranscription or expression of a heterologous polynucleotide in a cell,the method comprising introducing into a cell an isolated polynucleotidecomprising a manganese superoxide dismutase regulatory element, whereinthe regulatory element comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NO:5, SEQ NO:1 and nucleotide sequenceshaving at least about 90% identity to SEQ ID NO:1, the regulatoryelement being capable of causing inducible transcription or expressionof an operably linked heterologous polynucleotide.
 23. The method ofclaim 22 further comprising introducing into the cell an effectiveamount of a compound which activates the regulatory element to inducetranscription or expression of an operatively linked polynucleotide, ora polynucleotide encoding the compound.
 24. The method of claim 23wherein the compound is an inflammatory mediator.
 25. The method ofclaim 24 wherein the compound is selected from the group consisting ofTNF-α, IL-1β, and LPS.
 26. The method of claim 23 wherein the compoundis 5-aminosalicylic acid.
 27. The method of claim 22 wherein theregulatory element is operatively linked to a heterologouspolynucleotide.